Daniel Otzen's Publications List

Long-term-stable ether-lipid vs conventional ester-lipid bicelles in oriented solid-state NMR: altered structural information in studies of antimicrobial peptides.

2011-09-18T10:32:31Z

Recently, ether lipids have been introduced as long-term stable alternatives to the more natural, albeit easier degradable, ester lipids in the preparation of oriented lipid bilayers and bicelles for oriented-sample solid-state NMR spectroscopy. Here we report that ether lipids such as the frequently used 14-O-PC (1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) may induce significant changes in t...

Kresten Bertelsen, Brian Vad, Erik H Nielsen, Sara K Hansen, Troels Skrydstrup, Daniel E Otzen, Thomas Vosegaard, Niels Chr Nielsen (2011) J Phys Chem B 115: 8 1767-1774

SDS-facilitated in vitro formation of a transmembrane B-type cytochrome is mediated by changes in local pH.

2011-09-18T10:32:31Z

The folding and stabilization of α-helical transmembrane proteins are still not well understood. Following cofactor binding to a membrane protein provides a convenient method to monitor the formation of appropriate native structures. We have analyzed the assembly and stability of the transmembrane cytochrome b(559)', which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer ...

Mathias Weber, Alexander Prodöhl, Carolin Dreher, Christian Becker, Jarl Underhaug, Anna Sigrid Pii Svane, Anders Malmendal, Niels Chr Nielsen, Daniel Otzen, Dirk Schneider (2011) J Mol Biol 407: 4 594-606

Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation.

2011-09-18T10:32:31Z

One of the major hallmarks of Parkinson disease is aggregation of the protein α-synuclein (αSN). Aggregate cytotoxicity has been linked to an oligomeric species formed at early stages in the aggregation process. Here we follow the fibrillation process of αSN in solution over time using small angle X-ray scattering and resolve four major coexisting species in the fibrillation process, namely mon...

Lise Giehm, Dmitri I Svergun, Daniel E Otzen, Bente Vestergaard (2011) Proc Natl Acad Sci U S A 108: 8 3246-3251

Assays for α-synuclein aggregation.

2011-09-18T10:32:31Z

This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively ...

Lise Giehm, Nikolai Lorenzen, Daniel E Otzen (2011) Methods 53: 3 295-305

Mapping the folding pathway of the transmembrane protein DsbB by protein engineering.

2011-09-18T10:32:31Z

The four-helical transmembrane protein DsbB (disulfide bond reducing protein B) folds and unfolds reversibly in mixed anionic/non-ionic micelles, consisting of an unfolding intermediate I and a rate-limiting transition state (TS) between I and the denatured state D. Here, I describe the analysis of the folding behavior of 12 different alanine-scanning mutants of DsbB. For all mutants, TS is as com...

Daniel E Otzen (2011) Protein Eng Des Sel 24: 1-2 139-149

The role of membrane properties in Mistic folding and dimerisation.

2011-09-18T10:32:31Z

Membranes not only provide cellular compartmentalization but influence protein behavior and folding by virtue of the multitude of different lipid types. We have studied the impact of lipid composition on the folding of the membrane-associated protein Mistic from B. subtilis. We use dimerisation via the single Cys3 residue as monitor for the degree of correct folding, since mis- or unfolding will e...

Dilip K Debnath, Rajiv Vaid Basaiawmoit, Kåre Lehmann Nielsen, Daniel E Otzen (2011) Protein Eng Des Sel 24: 1-2 89-97

SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the Arg124His mutation.

2011-09-18T10:32:31Z

Human transforming growth factor β induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compr...

R V Basaiawmoit, C L P Oliveira, K Runager, C S Sørensen, M A Behrens, B-H Jonsson, T Kristensen, G K Klintworth, J J Enghild, J Skov Pedersen, D E Otzen (2011) J Mol Biol 408: 3 503-513

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

2011-09-18T10:32:31Z

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substi...

Kasper Runager, Rajiv V Basaiawmoit, Taru Deva, Maria Andreasen, Zuzana Valnickova, Charlotte S Sørensen, Henrik Karring, Ida B Thøgersen, Gunna Christiansen, Jarl Underhaug, Torsten Kristensen, Niels Chr Nielsen, Gordon K Klintworth, Daniel E Otzen, Jan J Enghild (2011) J Biol Chem 286: 7 4951-4958

Lactadherin binds to phosphatidylserine-containing vesicles in a two-step mechanism sensitive to vesicle size and composition.

2011-09-18T10:32:31Z

Lactadherin binds to phosphatidylserine (PS) in a stereospecific and calcium independent manner that is promoted by vesicle curvature. Because membrane binding of lactadherin is supported by a PS content of as little as 0.5%, lactadherin is a useful marker for cell stress where limited PS is exposed, as well as for apoptosis where PS freely traverses the plasma membrane. To gain further insight in...

Daniel E Otzen, Kristine Blans, Huabing Wang, Gary E Gilbert, Jan T Rasmussen (2011) Biochim Biophys Acta :

Protein-surfactant interactions: a tale of many states.

2011-09-18T10:32:31Z

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denat...

Daniel Otzen (2011) Biochim Biophys Acta 1814: 5 562-591

Mechanical Stress Affects Glucagon Fibrillation Kinetics and Fibril Structure.

2011-09-18T10:32:31Z

Mechanical stress can strongly influence the capability of a protein to aggregate and the kinetics of aggregation, but there is little insight into the underlying mechanism. Here we study the effect of different mechanical stress conditions on the fibrillation of the peptide hormone glucagon, which forms different fibrils depending on temperature, pH, ionic strength, and concentration. A combinati...

Francesca Macchi, Søren V Hoffmann, Martin Carlsen, Brian Vad, Alberto Imparato, Christian Rischel, Daniel E Otzen (2011) Langmuir :

Assembling good amyloid: some structures at last.

2011-09-18T10:32:31Z

Taylor et al. report the crystal structure of CsgC, a redox-active member of E. coli's curli-producing Csg operon. The outer membrane protein CsgG is a potential redox-substrate and might exist in an octameric structure with a periplasmatic CsgC binding site, highlighting a potential role for disulfide bonds in curli production.

Daniel E Otzen (2011) Structure 19: 9 1207-1209

Fibrillation of the Major Curli Subunit CsgA under a Wide Range of Conditions Implies a Robust Design of Aggregation.

2011-09-18T10:32:31Z

The amyloid fold is usually considered a result of protein misfolding. However, a number of studies have recently shown that the amyloid structure is also used in nature for functional purposes. CsgA is the major subunit of Escherichia coli curli, one of the most well-characterized functional amyloids. Here we show, using a highly efficient approach to prepare monomeric CsgA, that in vitro fibrill...

Morten S Dueholm, Søren B Nielsen, Kim L Hein, Poul Nissen, Matthew Chapman, Gunna Christiansen, Per Halkjær Nielsen, Daniel E Otzen (2011) Biochemistry :

Effect of protein-surfactant interactions on aggregation of β-lactoglobulin.

2011-09-18T10:32:31Z

The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alk...

Jon G Hansted, Peter L Wejse, Hans Bertelsen, Daniel E Otzen (2011) Biochim Biophys Acta 1814: 5 713-723

Aβ1-16 Can Aggregate and Induce the Production of Reactive Oxygen Species, Nitric Oxide, and Inflammatory Cytokines.

2011-09-18T10:32:31Z

Amyloid-β (Aβ40/42) aggregates containing the cross-β-sheet structure are associated with the pathogenesis of Alzheimer's disease (AD). It is generally accepted that the N-terminal peptide of Aβ40/42, Aβ1-16, does not aggregate, and is not cytotoxic. However, we here show that Aβ1-16 can aggregate, and form cytotoxic aggregates containing β-turns and regular non-amyloid β-sheet structures....

Xue-Ting Du, Li Wang, Yu-Jiong Wang, Maria Andreasen, Da-Wei Zhan, Ying Feng, Min Li, Min Zhao, Daniel Otzen, Di Xue, Yang Yang, Rui-Tian Liu (2011) J Alzheimers Dis :

The interaction of equine lysozyme:oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism.

2011-09-18T10:32:31Z

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to a...

Søren B Nielsen, Kristina Wilhelm, Brian Vad, Jürgen Schleucher, Ludmilla A Morozova-Roche, Daniel Otzen (2010) J Mol Biol 398: 2 351-361

Pardaxin permeabilizes vesicles more efficiently by pore formation than by disruption.

2011-09-18T10:32:31Z

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance (13)C solid-state nuclear magnetic resonance to provide a mo...

Brian S Vad, Kresten Bertelsen, Charlotte Hau Johansen, Jan Mondrup Pedersen, Troels Skrydstrup, Niels Chr Nielsen, Daniel E Otzen (2010) Biophys J 98: 4 576-585

beta-Sheet aggregation of kisspeptin-10 is stimulated by heparin but inhibited by amphiphiles.

2011-09-18T10:32:31Z

The murine 10-residue neurohormone kisspeptin (YNWNSFGLRY) is an important regulator of reproductive behavior and gonadotrophin secretion. It is known to form a random coil in solution, but undergoes a structural change in the presence of membranes although the nature of this change is not fully determined. The peptide's conformational versatility raises the question whether it is also able to for...

Søren B Nielsen, Magnus Franzmann, Rajiv V Basaiawmoit, Reinhard Wimmer, Jens D Mikkelsen, Daniel E Otzen (2010) Biopolymers 93: 8 678-689

Crystallographic analysis reveals a unique lidocaine binding site on human serum albumin.

2011-09-18T10:32:31Z

Human serum albumin (HSA), the major protein component in blood plasma and in extravascular spaces, is known to participate in the binding and transport of a variety of endogenous and exogenous organic compounds with anionic or electronegative features. We here report on the 3.3A resolution crystal structure of HSA complexed with the cationic, and widely used, anesthetic lidocaine. We find that li...

Kim Langmach Hein, Ulrich Kragh-Hansen, J Preben Morth, Martin D Jeppesen, Daniel Otzen, Jesper V Møller, Poul Nissen (2010) J Struct Biol 171: 3 353-360

In vitro association of fragments of a beta-sheet membrane protein.

2011-09-18T10:32:31Z

Although the beta-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25-35% of the fragment populations to no...

D Debnath, K L Nielsen, D E Otzen (2010) Biophys Chem 148: 1-3 112-120

Glucagon fibril polymorphism reflects differences in protofilament backbone structure.

2011-09-18T10:32:31Z

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the ...

Christian Beyschau Andersen, Matthew R Hicks, Valeria Vetri, Brian Vandahl, Henrik Rahbek-Nielsen, Henning Thøgersen, Ida Bukh Thøgersen, Jan Johannes Enghild, Louise C Serpell, Christian Rischel, Daniel Erik Otzen (2010) J Mol Biol 397: 4 932-946

Divorcing folding from function: how acylation affects the membrane-perturbing properties of an antimicrobial peptide.

2011-09-18T10:32:31Z

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an alpha-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilizatio...

Brian Vad, Line Aagot Thomsen, Kresten Bertelsen, Magnus Franzmann, Jan Mondrup Pedersen, Søren B Nielsen, Thomas Vosegaard, Zuzana Valnickova, Troels Skrydstrup, Jan J Enghild, Reinhard Wimmer, Niels Chr Nielsen, Daniel E Otzen (2010) Biochim Biophys Acta 1804: 4 806-820

Fast mapping of global protein folding states by multivariate NMR: a GPS for proteins.

2011-09-18T10:32:31Z

To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method that provides such an overview. GPS NMR exploits the unique ability of NMR to simultaneously record s...

Anders Malmendal, Jarl Underhaug, Daniel E Otzen, Niels C Nielsen (2010) PLoS One 5: 4

Proteomic changes in response to chromium(VI) toxicity in Pseudomonas aeruginosa.

2011-09-18T10:32:31Z

A proteomic approach was used to identify proteins involved in Cr(VI) stress response of Pseudomonas aeruginosa to toxic Cr(VI). Cytosolic and membrane fractions from bacteria exposed to 300 mg l(-1) Cr(VI) were prepared, 2D gel electrophoresis in combination with MALDI-TOF MS and LC-MS/MS was used to identify proteins whose expression level increased or decreased upon exposure to Cr(VI). Overexpr...

Nur Koçberber Kiliç, Allan Stensballe, Daniel Erik Otzen, Gönül Dönmez (2010) Bioresour Technol 101: 7 2134-2140

The role of protonation in protein fibrillation.

2011-09-18T10:32:31Z

Many proteins fibrillate at low pH despite a high population of charged side chains. Therefore exchange of protons between the fibrillating peptide and its surroundings may play an important role in fibrillation. Here, we use isothermal titration calorimetry to measure exchange of protons between buffer and the peptide hormone glucagon during fibrillation. Glucagon absorbs or releases protons to a...

Martin D Jeppesen, Peter Westh, Daniel E Otzen (2010) FEBS Lett 584: 4 780-784

The diversity of FtsY-lipid interactions.

2011-09-18T10:32:31Z

The bacterial signal recognition particle (SRP) receptor FtsY forms a complex with the SRP Ffh to target nascent polypeptide chains to the bacterial inner membrane. How FtsY interacts with lipids and associates to the membrane is unclear. Here, we show that vesicle binding leads to partial protection against proteolytic degradation and a change in secondary structure, which differs depending on wh...

M E Reinau, I B Thøgersen, J J Enghild, K L Nielsen, D E Otzen (2010) Biopolymers 93: 7 595-606

Impact of the antimicrobial peptide Novicidin on membrane structure and integrity.

2011-09-18T10:32:31Z

We have studied the impact of an 18-residue cationic antimicrobial peptide Novicidin (Nc) on the structure and integrity of partially anionic lipid membranes using oriented circular dichroism (OCD), quartz crystal microbalance with dissipation (QCM-D), dual polarization interferometry (DPI), calcein dye leakage and fluorescence spectroscopy. OCD consistently showed that Nc is bound in an alpha-hel...

Søren B Nielsen, Daniel E Otzen (2010) J Colloid Interface Sci :

Strategies to increase the reproducibility of protein fibrillization in plate reader assays.

2011-09-18T10:32:31Z

There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140-amino-acid protein...

Lise Giehm, Daniel E Otzen (2010) Anal Biochem 400: 2 270-281

Stop-and-go kinetics in amyloid fibrillation.

2011-09-18T10:32:31Z

Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions o...

Jesper Ferkinghoff-Borg, Jesper Fonslet, Christian Beyschau Andersen, Sandeep Krishna, Simone Pigolotti, Hisashi Yagi, Yuji Goto, Daniel Otzen, Mogens H Jensen (2010) Phys Rev E Stat Nonlin Soft Matter Phys 82: 1 Pt 1

Cell-free synthesis and folding of transmembrane OmpA reveals higher order structures and premature truncations.

2011-09-18T10:32:31Z

We use a cell-free transcription-translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain...

Dilip K Debnath, Daniel E Otzen (2010) Biophys Chem 152: 1-3 80-88

Functional amyloid: turning swords into plowshares.

2011-09-18T10:32:31Z

Evidence is growing at an increasing -pace that amyloid fibers are not just the result of aberrant protein folding associated with neurodegenerative diseases, but are widespread in nature for beneficial reasons. Amyloid is an attractive building material because its robust design and simple repetitive structure make for very durable and metabolically cheap material. But this requires that the prod...

Daniel Otzen (2010) Prion 4: 4 256-264

Amyloid structure--one but not the same: the many levels of fibrillar polymorphism.

2011-09-18T10:32:31Z

Many proteins and peptides can form amyloid-like structures both in vivo and in vitro. Although strikingly similar fibrillar structures can be observed across a variety of amino acid sequences, the fibrils formed often exhibit a stunning wealth of polymorphisms at the level of electron or atomic force microscopy. This appears to violate the Anfinsen principle seen for globular proteins, where each...

Jesper S Pedersen, Christian B Andersen, Daniel E Otzen (2010) FEBS J 277: 22 4591-4601

Amyloid formation in surfactants and alcohols: membrane mimetics or structural switchers?

2011-09-18T10:32:31Z

Attempts to understand the biophysical foundations and biochemical consequences of protein aggregation process are greatly aided by conditions which provide either robust and reliable reaction conditions or constitute mimics of the physiological conditions. While both anionic surfactants such as SDS and fluorinated alcohols such as TFE are often championed as membrane mimics in one way or another,...

Daniel E Otzen (2010) Curr Protein Pept Sci 11: 5 355-371

Membrane interactions of novicidin, a novel antimicrobial peptide: phosphatidylglycerol promotes bilayer insertion.

2011-09-18T10:32:31Z

Novicidin is an antimicrobial peptide derived from ovispirin, a cationic peptide which originated from the ovine cathelicidin SMAP-29. Novicidin, however, has been designed to minimize the cytotoxic properties of SMAP-29 and ovisipirin toward achieving potential therapeutic applications. We present an analysis of membrane interactions and lipid bilayer penetration of novicidin, using an array of b...

Jerzy Dorosz, Yana Gofman, Sofiya Kolusheva, Daniel Otzen, Nir Ben-Tal, Niels Chr Nielsen, Raz Jelinek (2010) J Phys Chem B 114: 34 11053-11060

Flexibility of the thrombin-activatable fibrinolysis inhibitor pro-domain enables productive binding of protein substrates.

2011-09-18T10:32:31Z

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, ...

Zuzana Valnickova, Laura Sanglas, Joan L Arolas, Steen V Petersen, Christine Schar, Daniel Otzen, Francesc X Aviles, F Xavier Gomis-Rüth, Jan J Enghild (2010) J Biol Chem 285: 49 38243-38250

An Aß concatemer with altered aggregation propensities.

2011-09-18T10:32:31Z

We present an analysis of the conformational and aggregative properties of an Aß concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Aß peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a...

L Giehm, F Dal Degan, P Fraser, S Klysner, Daniel E Otzen (2010) Biochim Biophys Acta 1804: 10 2025-2035

Occupancy of nonannular lipid binding sites on KcsA greatly increases the stability of the tetrameric protein.

2011-09-18T10:32:31Z

KcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as "nonannular" sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we used a thermal denaturation assay and detergent-phosp...

I Triano, F N Barrera, M L Renart, M L Molina, G Fernández-Ballester, J A Poveda, A M Fernández, J A Encinar, A V Ferrer-Montiel, D Otzen, J M González-Ros (2010) Biochemistry 49: 25 5397-5404

SDS-induced fibrillation of alpha-synuclein: an alternative fibrillation pathway.

2011-09-18T10:32:31Z

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably diff...

Lise Giehm, Cristiano Luis Pinto Oliveira, Gunna Christiansen, Jan Skov Pedersen, Daniel E Otzen (2010) J Mol Biol 401: 1 115-133

A thermodynamic analysis of fibrillar polymorphism.

2011-09-18T10:32:31Z

We explore the thermodynamic properties of three different fibrils of the peptide hormone glucagon, formed under different salt conditions (glycine, sulfate and NaCl, respectively), and differing considerably in compactness. The three fibrils display a large variation in the specific heat capacity DeltaC(p) determined by isothermal titration calorimetry. Sulfate fibrils show a negative DeltaC(p) e...

Martin D Jeppesen, Kim Hein, Poul Nissen, Peter Westh, Daniel E Otzen (2010) Biophys Chem 149: 1-2 40-46

Functional amyloid in Pseudomonas.

2011-09-18T10:32:31Z

Summary Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined...

Morten S Dueholm, Steen V Petersen, Mads Sønderkær, Poul Larsen, Gunna Christiansen, Kim L Hein, Jan J Enghild, Jeppe L Nielsen, Kåre L Nielsen, Per H Nielsen, Daniel E Otzen (2010) Mol Microbiol :

Branching in amyloid fibril growth.

2011-09-18T10:32:31Z

Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first tim...

Christian Beyschau Andersen, Hisashi Yagi, Mauro Manno, Vincenzo Martorana, Tadato Ban, Gunna Christiansen, Daniel Erik Otzen, Yuji Goto, Christian Rischel (2009) Biophys J 96: 4 1529-1536

The influence of vesicle size and composition on alpha-synuclein structure and stability.

2011-09-18T10:32:31Z

Monomeric alpha-synuclein (alphaSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in alpha-helix structure. Here we show that at physiological pH and ionic strength, alphaSN incubated with different anionic lipid vesicles undergoes a marked increase in alpha-helical content at a temperature dictated either by the temperature of...

Lars Kjaer, Lise Giehm, Thomas Heimburg, Daniel Otzen (2009) Biophys J 96: 7 2857-2870

A versatile approach to beta-amyloid fibril-binding compounds exploiting the Shirakawa/Hayashi protocol for trans-alkene synthesis.

2011-09-18T10:32:31Z

Application of the Sonogashira coupling reaction followed by a trans-selective alkyne reduction proved highly adaptable for the efficient synthesis of a class of beta-amyloid fibril binding compounds possessing a styrylbenzene motif such as FSB, an FSB dimer, and (19)F-BAY94-9172.

Tri H V Huynh, Mette Louise H Mantel, Katrine Mikkelsen, Anders T Lindhardt, Niels Chr Nielsen, Daniel Otzen, Troels Skrydstrup (2009) Org Lett 11: 4 999-1002

A SAXS study of glucagon fibrillation.

2011-09-18T10:32:31Z

Protein amyloid formation proceeds through a number of different stages. Oligomeric species observed at early stages have aroused particular interest because of evidence for their involvement in cytotoxic processes such as membrane permeabilization. It is unclear whether these oligomers are obligate precursors to fibrils or represent "dead-end" species that impede fibrillation. Because of the many...

Cristiano Luis Pinto Oliveira, Manja Annette Behrens, Jesper Søndergaard Pedersen, Kurt Erlacher, Daniel Otzen, Jan Skov Pedersen (2009) J Mol Biol 387: 1 147-161

Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan.

2011-09-18T10:32:31Z

The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been changed to serines to emulate the reduced peptide. SerADan aggregates rapidly at pH 5.0 and 7.5 in a serie...

Lise Nesgaard, Brian Vad, Gunna Christiansen, Daniel Otzen (2009) Biochim Biophys Acta 1794: 1 84-93

Alpha-Lactalbumin is unfolded by all classes of surfactants but by different mechanisms.

2011-09-18T10:32:31Z

We show that all four classes of surfactants (anionic, cationic, non-ionic, and zwitterionic) denature alpha-lactalbumin (alphaLA), making alphaLA an excellent model system to compare their denaturation mechanisms. This involves at least two steps in all surfactants but is more complex in charged surfactants due to their strong binding properties. At very low concentrations, charged surfactants bi...

Daniel E Otzen, Pankaj Sehgal, Peter Westh (2009) J Colloid Interface Sci 329: 2 273-283

Modeling proteasome dynamics in Parkinson's disease.

2011-09-18T10:32:31Z

In Parkinson's disease (PD), there is evidence that alpha-synuclein (alphaSN) aggregation is coupled to dysfunctional or overburdened protein quality control systems, in particular the ubiquitin-proteasome system. Here, we develop a simple dynamical model for the on-going conflict between alphaSN aggregation and the maintenance of a functional proteasome in the healthy cell, based on the premise t...

Kim Sneppen, Ludvig Lizana, Mogens H Jensen, Simone Pigolotti, Daniel Otzen (2009) Phys Biol 6: 3

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants.

2011-09-18T10:32:31Z

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the su...

Jonas Høeg Hansen, Steen Vang Petersen, Kell Kleiner Andersen, Jan J Enghild, Ture Damhus, Daniel Otzen (2009) Biopolymers 91: 3 221-231

Unique identification of supramolecular structures in amyloid fibrils by solid-state NMR spectroscopy.

2011-09-18T10:32:31Z

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 A resolution. Symmetry information contained in the easily obtainable resonance assignments from solid-state NMR spectra (see picture), along with long-range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

Jakob T Nielsen, Morten Bjerring, Martin D Jeppesen, Ronnie O Pedersen, Jan M Pedersen, Kim L Hein, Thomas Vosegaard, Troels Skrydstrup, Daniel E Otzen, Niels C Nielsen (2009) Angew Chem Int Ed Engl 48: 12 2118-2121

Widespread abundance of functional bacterial amyloid in mycolata and other gram-positive bacteria.

2011-09-18T10:32:31Z

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six ...

Peter Bruun Jordal, Morten Simonsen Dueholm, Poul Larsen, Steen Vang Petersen, Jan Johannes Enghild, Gunna Christiansen, Peter Højrup, Per Halkjaer Nielsen, Daniel Erik Otzen (2009) Appl Environ Microbiol 75: 12 4101-4110

How chain length and charge affect surfactant denaturation of acyl coenzyme A binding protein (ACBP).

2011-09-18T10:32:31Z

Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the den...

Kell K Andersen, Daniel E Otzen (2009) J Phys Chem B 113: 42 13942-13952

Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids.

2011-09-18T10:32:31Z

The cytosolic protein Ffh transports membrane proteins from the ribosome to the inner membrane in complex with 4.5S RNA. Here we show that native Ffh binds to the hydrophobic probe ANS in a 1 Ffh:3 ANS stoichiometry, revealing a hydrophobic binding site. Thermal precipitation of Ffh is shifted upwards by approximately 10 degrees C by ANS or substrate protein, suggesting that the hydrophobic bindin...

Marika E Reinau, Daniel E Otzen (2009) Arch Biochem Biophys 492: 1-2 48-53

Influence of tunable external stimuli on the self-assembly of guanosine supramolecular nanostructures studied by atomic force microscope.

2011-09-18T10:32:31Z

The self-assembly of guanosine (G) molecules on solid surfaces is investigated by tapping-mode atomic force microscopy (AFM) upon controlling and introducing external factors (stimuli) to the G stock solution such as incubation time, presence/absence of metal cations, and mechanical shaking. Surprisingly, at different stages of incubation time at room temperature and in the absence of any metal ca...

Yinli Li, Mingdong Dong, Daniel E Otzen, Yuheng Yao, Bo Liu, Flemming Besenbacher, Wael Mamdouh (2009) Langmuir 25: 23 13432-13437

Synthesis of a ketomethylene isostere of the fibrillating peptide SNNFGAILSS.

2011-09-18T10:32:31Z

The direct synthesis of a ketomethylene isostere of the fibril-forming decapeptide SNNFGAILSS is presented with the goal of understanding how small structural changes alter the ability of such peptides to recognize each other for beta-sheet formation. The key synthetic step relies on a SmI(2)-mediated coupling of a N-tetrapeptidyl oxazolidinone with a simple acrylate followed by deprotection of th...

Tina Mittag, Daniel E Otzen, Niels Chr Nielsen, Troels Skrydstrup (2009) J Org Chem 74: 20 7955-7957

Interaction of a potyviral VPg with anionic phospholipid vesicles.

2011-09-18T10:32:31Z

The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC...

Kimmo I Rantalainen, Peter A Christensen, Anders Hafrén, Daniel E Otzen, Nisse Kalkkinen, Kristiina Mäkinen (2009) Virology 395: 1 114-120

The role of decorated SDS micelles in sub-CMC protein denaturation and association.

2011-09-18T10:32:31Z

We have combined spectroscopy, chromatography, calorimetry, and small-angle X-ray scattering (SAXS) to provide a comprehensive structural and stoichiometric description of the sodium dodecyl sulfate (SDS)-induced denaturation of the 86-residue alpha-helical bovine acyl-coenzyme-A-binding protein (ACBP). Denaturation is a multistep process. Initial weak binding of 1-3 SDS molecules per protein mole...

Kell K Andersen, Cristiano L Oliveira, Kim L Larsen, Flemming M Poulsen, Thomas H Callisen, Peter Westh, Jan S Pedersen, Daniel Otzen (2009) J Mol Biol 391: 1 207-226

Surfactant protein SP-B strongly modifies surface collapse of phospholipid vesicles: insights from a quartz crystal microbalance with dissipation.

2011-09-18T10:32:31Z

Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer f...

Elisa J Cabré, Jenny Malmström, Duncan Sutherland, J Pérez-Gil, Daniel E Otzen (2009) Biophys J 97: 3 768-776

Quantitative use of paramagnetic relaxation enhancements for determining orientations and insertion depths of peptides in micelles.

2011-09-18T10:32:31Z

We describe the background and implementation of a method to determine, at atomic resolution, the insertion depths and orientations of peptides embedded in micelles. A nonperturbing paramagnetic agent--Gd(DTPA-BMA)--was used to induce paramagnetic relaxation enhancements (PREs) of peptide atoms inside the micelle. By calibrating these PREs it was possible to translate them into distance restraints...

Magnus Franzmann, Daniel Otzen, Reinhard Wimmer (2009) Chembiochem 10: 14 2339-2347

Aggregation of S6 in a quasi-native state by sub-micellar SDS.

2011-09-18T10:32:31Z

Anionic surfaces promote protein fibrillation in vitro and in vivo. Monomeric SDS has also been shown to stimulate this process. We describe the dynamics of conformational changes and aggregative properties of the model protein S6 at sub-micellar SDS concentrations. S6 exhibits a rich and pH-sensitive diversity in conformational changes around 0.2-2 mM SDS, in which several transitions occur over ...

Daniel E Otzen, Lise W Nesgaard, Kell K Andersen, Jonas Høeg Hansen, Gunna Christiansen, Hidekazu Doe, Pankaj Sehgal (2008) Biochim Biophys Acta 1784: 2 400-414

Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions.

2011-09-18T10:32:31Z

Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-amin...

Lise Giehm, Casper Christensen, Ulrik Boas, Peter M H Heegaard, Daniel E Otzen (2008) Biopolymers 89: 6 522-529

Amyloid-like adhesins produced by floc-forming and filamentous bacteria in activated sludge.

2011-09-18T10:32:31Z

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abund...

Poul Larsen, Jeppe Lund Nielsen, Daniel Otzen, Per Halkjaer Nielsen (2008) Appl Environ Microbiol 74: 5 1517-1526

Amyloid-a state in many guises: survival of the fittest fibril fold.

2011-09-18T10:32:31Z

Under appropriate conditions, essentially all proteins are able to aggregate to form long, well-ordered and beta-sheet-rich arrays known as amyloid-like fibrils. These fibrils consist of varying numbers of intertwined protofibrils and can for any given protein exhibit a wealth of different forms at the ultrastructural level. Traditionally, this structural variability or polymorphism has been attri...

Jesper S Pedersen, Daniel E Otzen (2008) Protein Sci 17: 1 2-10

Thermodynamics and mechanism of cutinase stabilization by trehalose.

2011-09-18T10:32:31Z

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that l...

Ricardo P Baptista, Shona Pedersen, Gonçalo J M Cabrita, Daniel E Otzen, Joaquim M S Cabral, Eduardo P Melo (2008) Biopolymers 89: 6 538-547

We find them here, we find them there: functional bacterial amyloid.

2011-09-18T10:32:31Z

Protein amyloid is often deposited in connection with neurodegenerative diseases. Such deposits generally possess three principal drawbacks: cytotoxicity, lack of spatial control in their deposition and structural polymorphism. These are typical features of biologically non-optimized systems which have not been exposed to evolutionary pressure. Nevertheless, Nature uses the cross-beta self-organiz...

D Otzen, P H Nielsen (2008) Cell Mol Life Sci 65: 6 910-927

Global study of myoglobin-surfactant interactions.

2011-09-18T10:32:31Z

Surfactants interact with proteins in multifarious ways which depend on surfactant concentration and structure. To obtain a global overview of this process, we have analyzed the interaction of horse myoglobin (Mb) with an anionic (SDS) and cationic (CTAC) surfactant, using both equilibrium titration techniques and stopped-flow kinetics. Binding and kinetics of conformational changes can be divided...

Kell K Andersen, Peter Westh, Daniel E Otzen (2008) Langmuir 24: 2 399-407

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism.

2011-09-18T10:32:31Z

Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we observe a low-wavelength band in the range 130-160 nm, whose intensity and peak position is sensitive to t...

Lise W Nesgaard, Søren V Hoffmann, Christian Beyschau Andersen, Anders Malmendal, Daniel E Otzen (2008) Biopolymers 89: 9 779-795

An unusual intrinsically disordered protein from the model legume Lotus japonicus stabilizes proteins in vitro.

2011-09-18T10:32:31Z

Intrinsic structural disorder is a prevalent feature of proteins with chaperone activity. Using a complementary set of techniques, we have structurally characterized LjIDP1 (intrinsically disordered protein 1) from the model legume Lotus japonicus, and our results provide the first structural characterization of a member of the Lea5 protein family (PF03242). Contrary to in silico predictions, we s...

Svend Haaning, Simona Radutoiu, Søren V Hoffmann, Jens Dittmer, Lise Giehm, Daniel E Otzen, Jens Stougaard (2008) J Biol Chem 283: 45 31142-31152

AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptide glucagon.

2011-09-18T10:32:31Z

We report on the mechanical characterization of individual mature amyloid fibrils by atomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy (SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptide hormone glucagon, were found to display a reversible elastic behaviour. Based on AFM morphology and SMFS studies, we suggest that the observed elast...

Mingdong Dong, Mads Bruun Hovgaard, Wael Mamdouh, Sailong Xu, Daniel Erik Otzen, Flemming Besenbacher (2008) Nanotechnology 19: 38

Differential adsorption of variants of the Thermomyces lanuginosus lipase on a hydrophobic surface suggests a role for local flexibility.

2011-09-18T10:32:31Z

Lipases are activated at interfaces between aqueous and hydrophobic phases, where they typically undergo conformational changes leading to significant activity increase. Here I use a quartz crystal microbalance with dissipation (QCM-D) to study changes in layer thickness and viscosity during the adsorption of variants of the Thermomyces lanuginosus lipase (TlL) onto a methyl-terminated hydrophobic...

Daniel Otzen (2008) Colloids Surf B Biointerfaces 64: 2 223-228

Spectroscopic evidence for the existence of an obligate pre-fibrillar oligomer during glucagon fibrillation.

2011-09-18T10:32:31Z

The 29-residue peptide hormone glucagon has been used as a model system for the study of amyloid-like fibrils. Atomic force microscopy (AFM) studies have detected putative oligomeric species during this lag phase, but this has not been confirmed by any spectroscopic technique. Here we use an attached pyrene group to detect association (excimer formation) between individual glucagon molecules. Our ...

Peter Astrup Christensen, Jesper Søndergaard Pedersen, Gunna Christiansen, Daniel Erik Otzen (2008) FEBS Lett 582: 9 1341-1345

Early stages of amyloid fibril formation studied by liquid-state NMR: the peptide hormone glucagon.

2011-09-18T10:32:31Z

The 29-residue peptide hormone glucagon forms amyloid fibrils within a few hours at low pH. In this study, we use glucagon as a model system to investigate fibril formation by liquid-state (1)H-NMR spectroscopy One-dimensional, correlation, and diffusion experiments monitoring the fibril formation process provide insight into the early stages of the pathway on which the molecules aggregate to fibr...

Anna Sigrid Pii Svane, Kasper Jahn, Taru Deva, Anders Malmendal, Daniel Erik Otzen, Jens Dittmer, Niels Chr Nielsen (2008) Biophys J 95: 1 366-377

Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation.

2011-09-18T10:32:31Z

Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound beta-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present s...

Stine K Knudsen, Allan Stensballe, Magnus Franzmann, Uffe B Westergaard, Daniel E Otzen (2008) Biochem J 412: 3 563-577

Amyloid adhesins are abundant in natural biofilms.

2011-09-18T10:32:31Z

Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with flu...

Poul Larsen, Jeppe Lund Nielsen, Morten Simonsen Dueholm, Ronald Wetzel, Daniel Otzen, Per Halkjaer Nielsen (2007) Environ Microbiol 9: 12 3077-3090

Changes in structures of milk proteins upon photo-oxidation.

2011-09-18T10:32:31Z

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, a...

Trine K Dalsgaard, Daniel Otzen, Jacob H Nielsen, Lotte B Larsen (2007) J Agric Food Chem 55: 26 10968-10976

Modulation of cutinase stability and structure by phospholipid detergents.

2011-09-18T10:32:31Z

Fusarium solani pisi cutinase hydrolyses triglycerides of different lengths. Here we show that micelle-forming short-chain (C6-C9) phospholipids significantly reduce cutinase stability (both below and above the critical micelle concentration cmc) and rates of folding (only above cmc), trapping cutinase in an inactive state which only regains activity over hours to days, rather than the few seconds...

Pankaj Sehgal, Søren Bang Nielsen, Shona Pedersen, Reinhard Wimmer, Daniel E Otzen (2007) Biochim Biophys Acta 1774: 12 1544-1554

Unfolding of beta-sheet proteins in SDS.

2011-09-18T10:32:31Z

Beta-sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two beta-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds ar...

Mette M Nielsen, Kell K Andersen, Peter Westh, Daniel E Otzen (2007) Biophys J 92: 10 3674-3685

The major allergen from birch tree pollen, Bet v 1, binds and permeabilizes membranes.

2011-09-18T10:32:31Z

The 159 residue Bet v 1 is the major allergen from birch tree pollen. Its natural function is unknown although it is capable of binding several types of physiologically relevant ligands in a centrally placed cavity in the protein structure. Here we use circular dichroism and fluorescence spectroscopy to show that Bet v 1 binds to DOPC and DOPG phospholipid vesicles in a pH-dependent manner. Bindin...

Jesper E Mogensen, Mercedes Ferreras, Reinhard Wimmer, Steen V Petersen, Jan J Enghild, Daniel E Otzen (2007) Biochemistry 46: 11 3356-3365

Aggregation as the basis for complex behaviour of cutinase in different denaturants.

2011-09-18T10:32:31Z

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equi...

Daniel E Otzen, Lise Giehm, Ricardo P Baptista, Søren R Kristensen, Eduardo P Melo, Shona Pedersen (2007) Biochim Biophys Acta 1774: 2 323-333

Heparin binding induces a conformational change in pigment epithelium-derived factor.

2011-09-18T10:32:31Z

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J....

Zuzana Valnickova, Steen V Petersen, Søren B Nielsen, Daniel E Otzen, Jan J Enghild (2007) J Biol Chem 282: 9 6661-6667

Quartz crystal microbalance studies of multilayer glucagon fibrillation at the solid-liquid interface.

2011-09-18T10:32:31Z

We have used a quartz crystal microbalance with dissipation (QCM-D) to monitor the changes in layer thickness and viscoelastic properties accompanying multilayer amyloid deposition in situ for the first time. By means of atomic force microscope imaging, an unequivocal correlation is established between the interfacial nucleation and growth of glucagon fibrils and the QCM-D response. The combinatio...

Mads Bruun Hovgaard, Mingdong Dong, Daniel Erik Otzen, Flemming Besenbacher (2007) Biophys J 93: 6 2162-2169

Alternative membrane protein conformations in alcohols.

2011-09-18T10:32:31Z

Alcohols modulate the oligomerization of membrane proteins in lipid bilayers. This can occur indirectly by redistributing lateral membrane pressure in a manner which correlates with alcohol hydrophobicity. Here we investigate the direct impact of different alcohol-water mixtures on membrane protein stability and solubility, using the two detergent-solubilized alpha-helical membrane proteins DsbB a...

D E Otzen, P Sehgal, L W Nesgaard (2007) Biochemistry 46: 14 4348-4359

p25alpha relocalizes in oligodendroglia from myelin to cytoplasmic inclusions in multiple system atrophy.

2011-09-18T10:32:31Z

p25alpha is an oligodendroglial protein that can induce aggregation of alpha-synuclein and accumulates in oligodendroglial cell bodies containing fibrillized alpha-synuclein in the neurodegenerative disease multiple system atrophy (MSA). We demonstrate biochemically that p25alpha is a constituent of myelin and a high-affinity ligand for myelin basic protein (MBP), and in situ immunohistochemistry ...

Yun Ju C Song, Ditte M S Lundvig, Yue Huang, Wei Ping Gai, Peter C Blumbergs, Peter Højrup, Daniel Otzen, Glenda M Halliday, Poul H Jensen (2007) Am J Pathol 171: 4 1291-1303

Dendrimer effects on peptide and protein fibrillation.

2011-09-18T10:32:31Z

Dendrimers are synthetic, symmetrically branched polymers that can be manufactured to a high degree of definition and therefore present themselves as monodisperse entities. Flexible and globular in shape and compartementalized into a partly inaccessible interior and a highly exposed surface, they offer numerous possibilities for interactions with and responses to biological macromolecules and bios...

Peter M H Heegaard, Ulrik Boas, Daniel E Otzen (2007) Macromol Biosci 7: 8 1047-1059

Aggregation and fibrillation of bovine serum albumin.

2011-09-18T10:32:31Z

The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a ...

Nikolaj K Holm, Stine K Jespersen, Lise V Thomassen, Tine Y Wolff, Pankaj Sehgal, Line A Thomsen, Gunna Christiansen, Christian Beyschau Andersen, Anders D Knudsen, Daniel E Otzen (2007) Biochim Biophys Acta 1774: 9 1128-1138

Glucagon amyloid-like fibril morphology is selected via morphology-dependent growth inhibition.

2011-09-18T10:32:31Z

The 29-residue peptide hormone glucagon readily fibrillates at low pH, but the structure and morphology of the fibrils are very sensitive to the environmental conditions. Here we have investigated the mechanism behind the differences in morphology observed when glucagon fibrils are formed at different peptide concentrations. Electron microscopy shows that fibrils formed at low glucagon concentrati...

Christian Beyschau Andersen, Daniel Otzen, Gunna Christiansen, Christian Rischel (2007) Biochemistry 46: 24 7314-7324

Compaction of ribosomal protein S6 by sucrose occurs only under native conditions.

2011-09-18T10:32:31Z

The effect of osmolyte sucrose on the stability and compaction of the folded and unfolded states of ribosomal protein S6 from Thermus thermophilus was analyzed. Confirming previous results obtained with sodium sulfate and trehalose, refolding stopped-flow measurements of S6 show that sucrose favors the conversion of the unfolded state ensemble to a highly compact structure (75% as compact as the f...

LuYang Chen, José A B Ferreira, Sílvia M B Costa, Gonçalo J M Cabrita, Daniel E Otzen, Eduardo Pinho Melo (2006) Biochemistry 45: 7 2189-2199

Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids.

2011-09-18T10:32:31Z

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by li...

Reinhard Wimmer, Kell K Andersen, Brian Vad, Mads Davidsen, Søren Mølgaard, Lise W Nesgaard, Hans H Kristensen, Daniel E Otzen (2006) Biochemistry 45: 2 481-497

The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting.

2011-09-18T10:32:31Z

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stabili...

Jesper Søndergaard Pedersen, Dantcho Dikov, James L Flink, Hans Aage Hjuler, Gunna Christiansen, Daniel Erik Otzen (2006) J Mol Biol 355: 3 501-523

The stability of transmembrane helix interactions measured in a biological membrane.

2011-09-18T10:32:31Z

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix in...

Carmen Finger, Thomas Volkmer, Alexander Prodöhl, Daniel E Otzen, Donald M Engelman, Dirk Schneider (2006) J Mol Biol 358: 5 1221-1228

Sulfates dramatically stabilize a salt-dependent type of glucagon fibrils.

2011-09-18T10:32:31Z

Recent work suggests that protein fibrillation mechanisms and the structure of the resulting protein fibrils are very sensitive to environmental conditions such as temperature and ionic strength. Here we report the effect of several inorganic salts on the fibrillation of glucagon. At acidic pH, fibrillation is much less influenced by cations than anions, for which the effects follow the electrosel...

Jesper Søndergaard Pedersen, James M Flink, Dantcho Dikov, Daniel Erik Otzen (2006) Biophys J 90: 11 4181-4194

Thermodynamics of unfolding of an integral membrane protein in mixed micelles.

2011-09-18T10:32:31Z

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl maltoside facilitate reversible and quantitative unfolding and refolding. The 4-transmembrane helix pro...

Pankaj Sehgal, Daniel E Otzen (2006) Protein Sci 15: 4 890-899

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS.

2011-09-18T10:32:31Z

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes,...

Shona Pedersen, Lise Nesgaard, Ricardo P Baptista, Eduardo P Melo, Søren R Kristensen, Daniel E Otzen (2006) Biopolymers 83: 6 619-629

N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon.

2011-09-18T10:32:31Z

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic re...

Jesper Søndergaard Pedersen, Dancho Dikov, Daniel Erik Otzen (2006) Biochemistry 45: 48 14503-14512

AFM study of glucagon fibrillation via oligomeric structures resulting in interwoven fibrils.

2011-09-18T10:32:31Z

Glucagon is a 29-residue amphiphatic hormone involved in the regulation of blood glucose levels in conjunction with insulin. In concentrated aqueous solutions, glucagon spontaneously aggregates to form amyloid fibrils, destroying its biological activity. In this study we utilize the atomic force microscope (AFM) to elucidate the fibrillation mechanism of glucagon at the nanoscale under acidic cond...

Mingdong Dong, Mads Bruun Hovgaard, Sailong Xu, Daniel Erik Otzen, Flemming Besenbacher (2006) Nanotechnology 17: 16 4003-4009

p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies.

2011-09-18T10:32:31Z

Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory ...

Evo Lindersson, Ditte Lundvig, Christine Petersen, Peder Madsen, Jens R Nyengaard, Peter Højrup, Torben Moos, Daniel Otzen, Wei-Ping Gai, Peter C Blumbergs, Poul Henning Jensen (2005) J Biol Chem 280: 7 5703-5715

p25alpha is flexible but natively folded and binds tubulin with oligomeric stoichiometry.

2011-09-18T10:32:31Z

p25alpha is a 219-residue protein which stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25alpha has been proposed to be natively unfolded, so that fol...

Daniel E Otzen, Ditte M S Lundvig, Reinhard Wimmer, Lotte H Nielsen, Jakob R Pedersen, Poul H Jensen (2005) Protein Sci 14: 6 1396-1409

Protein drug stability: a formulation challenge.

2011-09-18T10:32:31Z

The increasing use of recombinantly expressed therapeutic proteins in the pharmaceutical industry has highlighted issues such as their stability during long-term storage and means of efficacious delivery that avoid adverse immunogenic side effects. Controlled chemical modifications, such as substitutions, acylation and PEGylation, have fulfilled some but not all of their promises, while hydrogels ...

Sven Frokjaer, Daniel E Otzen (2005) Nat Rev Drug Discov 4: 4 298-306

Barriers to folding of the transmembrane domain of the Escherichia coli autotransporter adhesin involved in diffuse adherence.

2011-09-18T10:32:31Z

Adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated in the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to...

Jesper E Mogensen, Damini Tapadar, M Alexander Schmidt, Daniel E Otzen (2005) Biochemistry 44: 11 4533-4545

Conformational detours during folding of a collapsed state.

2011-09-18T10:32:31Z

The protein S6 is a useful model to probe the role of partially folded states in the folding process. In the absence of salt, S6 folds from the denatured state D to the native state N without detectable intermediates. High concentrations of sodium sulfate induce the accumulation of a collapsed state C, which is off the direct folding route. However, the mutation VA85 enables S6 to fold from C dire...

Daniel E Otzen (2005) Biochim Biophys Acta 1750: 2 146-153

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents.

2011-09-18T10:32:31Z

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginos...

Jesper E Mogensen, Pankaj Sehgal, Daniel E Otzen (2005) Biochemistry 44: 5 1719-1730

Stabilization of the ribosomal protein S6 by trehalose is counterbalanced by the formation of a putative off-pathway species.

2011-09-18T10:32:31Z

The effect of trehalose on folding and stability of the small ribosomal protein S6 was studied. Non-disruptive point mutations distributed along the protein structure were analyzed to characterize the stabilizing effect of trehalose and map the folding pathway of S6. On average, the stability of the wild-type and S6 mutants increases by 3 kcal/mol M trehalose. Despite the non-specific thermodynami...

Luyang Chen, Gonçalo J M Cabrita, Daniel E Otzen, Eduardo Pinho Melo (2005) J Mol Biol 351: 2 402-416

Interactions between folding factors and bacterial outer membrane proteins.

2011-09-18T10:32:31Z

The outer membrane is the first line of contact between Gram-negative bacteria and their external environment. Embedded in the outer membrane are integral outer membrane proteins (OMPs) that perform a diverse range of tasks. OMPs are synthesized in the cytoplasm and are translocated across the inner membrane and probably diffuse through the periplasm before they are inserted into the outer membran...

Jesper E Mogensen, Daniel E Otzen (2005) Mol Microbiol 57: 2 326-346

Misfolding of a bacterial autotransporter.

2011-09-18T10:32:31Z

The adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated into the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predic...

Jesper E Mogensen, Jörg H Kleinschmidt, M Alexander Schmidt, Daniel E Otzen (2005) Protein Sci 14: 11 2814-2827

Antagonism, non-native interactions and non-two-state folding in S6 revealed by double-mutant cycle analysis.

2011-09-18T10:32:31Z

When folding to the native state N in the presence of salt, the apparent two-state folder S6 transiently forms a transient off-pathway state C with substantial secondary and tertiary structure. Fifteen double mutant cycles were analysed to compare side-chain interaction energies DeltaDeltaG(int) in C, N and TS (the transition state between N and the denatured state). The kinetic signatures of thes...

Daniel Otzen (2005) Protein Eng Des Sel 18: 11 547-557

Using micellar mole fractions to assess membrane protein stability in mixed micelles.

2011-09-18T10:32:31Z

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations betwee...

P Sehgal, J E Mogensen, D E Otzen (2005) Biochim Biophys Acta 1716: 1 59-68

Modulation of S6 fibrillation by unfolding rates and gatekeeper residues.

2011-09-18T10:32:31Z

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, ...

Jesper Søndergaard Pedersen, Gunna Christensen, Daniel Erik Otzen (2004) J Mol Biol 341: 2 575-588

Investigating porcine pancreatic phospholipase A2 action on vesicles and supported planar bilayers using a quartz crystal microbalance with dissipation.

2011-09-18T10:32:31Z

We present an investigation of the activity of porcine pancreatic phospholipase A2 towards phospholipids. The phospholipids are presented in three different ways, namely as tethered vesicles, intact surface-bound vesicles, and supported planar bilayers (SPBs). The process is followed using a quartz crystal microbalance which measures both the frequency shift and the energy dissipation factor. This...

Pernille H Justesen, Tina Kristensen, Tony Ebdrup, Daniel Otzen (2004) J Colloid Interface Sci 279: 2 399-409

Transient formation of nano-crystalline structures during fibrillation of an Abeta-like peptide.

2011-09-18T10:32:31Z

During the first few minutes of fibrillation of a 14-residue peptide homologous to the hydrophobic C-terminal part of the Abeta-peptide, EM micrographs reveal small crystalline areas (100 to 150 nm, repeating unit 47 A) scattered in more amorphous material. On a longer time scale, these crystalline areas disappear and are replaced by tangled clusters resembling protofilaments (hours), and eventual...

Daniel E Otzen, Mikael Oliveberg (2004) Protein Sci 13: 5 1417-1421

Elimination of a misfolded folding intermediate by a single point mutation.

2011-09-18T10:32:31Z

We present an analysis of the folding behavior of the 159-residue major birch pollen allergen Bet v 1. The protein contains a water-filled channel running through it. Consequently, the protein has a hydrophobic shell, rather than a hydrophobic core. During the folding of the protein from either the urea-, pH-, or SDS-denatured state, Bet v 1 transiently populates a partially folded intermediate st...

Jesper E Mogensen, Henrik Ipsen, Jens Holm, Daniel E Otzen (2004) Biochemistry 43: 12 3357-3367

Transient aggregation and stable dimerization induced by introducing an Alzheimer sequence into a water-soluble protein.

2011-09-18T10:32:31Z

Transient contacts between denatured polypeptide chains are likely to play an important part in the initial stages of protein aggregation and fibrillation. To analyze the nature of such contacts, we have carried out a protein engineering study of the 102-residue protein U1A, which aggregates transiently in the wild-type form during refolding from the guanidinium chloride-denatured state. We have p...

Daniel E Otzen, Simona Miron, Mikael Akke, Mikael Oliveberg (2004) Biochemistry 43: 41 12964-12978

Correspondence between anomalous m- and DeltaCp-values in protein folding.

2011-09-18T10:32:31Z

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kin...

Daniel E Otzen, Mikael Oliveberg (2004) Protein Sci 13: 12 3253-3263

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein.

2011-09-18T10:32:31Z

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a ...

Daniel E Otzen (2003) J Mol Biol 330: 4 641-649

Trehalose favors a cutinase compact intermediate off-folding pathway.

2011-09-18T10:32:31Z

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the fold...

Eduardo P Melo, LuYang Chen, Joaquim M S Cabral, Peter Fojan, Steffen B Petersen, Daniel E Otzen (2003) Biochemistry 42: 24 7611-7617

Structural background of cyclodextrin-protein interactions.

2011-09-18T10:32:31Z

Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological...

F L Aachmann, D E Otzen, K L Larsen, R Wimmer (2003) Protein Eng 16: 12 905-912

Conformational plasticity in folding of the split beta-alpha-beta protein S6: evidence for burst-phase disruption of the native state.

2011-09-18T10:32:31Z

An increasing number of folding studies of two-state proteins shows that point mutations sometimes change the kinetic m-values, leading to kinks and curves in the chevron plots. The molecular origin of these changes is yet unclear although it is speculated that they are linked to structural rearrangement of the transition state or to accumulation of meta-stable intermediates. To shed more light on...

Daniel E Otzen, Mikael Oliveberg (2002) J Mol Biol 317: 4 613-627

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands.

2011-09-18T10:32:31Z

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this ...

Jesper E Mogensen, Reinhard Wimmer, Jørgen N Larsen, Michael D Spangfort, Daniel E Otzen (2002) J Biol Chem 277: 26 23684-23692

Burst-phase expansion of native protein prior to global unfolding in SDS.

2011-09-18T10:32:31Z

Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein enginee...

Daniel E Otzen, Mikael Oliveberg (2002) J Mol Biol 315: 5 1231-1240

Structural basis for cyclodextrins' suppression of human growth hormone aggregation.

2011-09-18T10:32:31Z

Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However,...

Daniel Erik Otzen, Benjamin Raerup Knudsen, Finn Aachmann, Kim Lambertsen Larsen, Reinhard Wimmer (2002) Protein Sci 11: 7 1779-1787

Protein unfolding in detergents: effect of micelle structure, ionic strength, pH, and temperature.

2011-09-18T10:32:31Z

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent inc...

Daniel E Otzen (2002) Biophys J 83: 4 2219-2230

Directed evolution of barnase stability using proteolytic selection.

2011-09-18T10:32:31Z

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistanc...

Jesper S Pedersen, Daniel E Otzen, Peter Kristensen (2002) J Mol Biol 323: 1 115-123

Folding of circular permutants with decreased contact order: general trend balanced by protein stability.

2011-09-18T10:32:31Z

To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small beta-alpha-beta protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log k(f)) with decreasing contact order. At th...

M O Lindberg, J Tångrot, D E Otzen, D A Dolgikh, A V Finkelstein, M Oliveberg (2001) J Mol Biol 314: 4 891-900

A simple way to measure protein refolding rates in water.

2011-09-18T10:32:31Z

Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be p...

D E Otzen, M Oliveberg (2001) J Mol Biol 313: 3 479-483

The spectral and thermodynamic properties of staphylococcal enterotoxin A, E, and variants suggest that structural modifications are important to control their function.

2011-09-18T10:32:31Z

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally sta...

A Cavallin, H Arozenius, K Kristensson, P Antonsson, D E Otzen, P Björk, G Forsberg (2000) J Biol Chem 275: 3 1665-1672

Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems.

2011-09-18T10:32:31Z

For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylen...

H Umakoshi, J Persson, M Kroon, H O Johansson, D E Otzen, R Kuboi, F Tjerneld (2000) J Chromatogr B Biomed Sci Appl 743: 1-2 13-19

Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly.

2011-09-18T10:32:31Z

Limited solubility and precipitation of amyloidogenic sequences such as the Alzheimer peptide (beta-AP) are major obstacles to a molecular understanding of protein fibrillation and deposition processes. Here we have circumvented the solubility problem by stepwise engineering a beta-AP homology into a soluble scaffold, the monomeric protein S6. The S6 construct with the highest beta-AP homology cry...

D E Otzen, O Kristensen, M Oliveberg (2000) Proc Natl Acad Sci U S A 97: 18 9907-9912

Structural changes in the transition state of protein folding: alternative interpretations of curved chevron plots.

2011-09-18T10:32:31Z

The interpretation of folding rates is often rationalized within the context of transition state theory. This means that the reaction rate is linked to an activation barrier, the height of which is determined by the free energy difference between a ground state (the starting point) and an apparent transition state. Changes in the folding kinetics are thus caused by effects on either the ground sta...

D E Otzen, O Kristensen, M Proctor, M Oliveberg (1999) Biochemistry 38: 20 6499-6511

Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects.

2011-09-18T10:32:31Z

Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that th...

D E Otzen, A R Fersht (1999) Protein Eng 12: 1 41-45

Electrostatics in the active site of an alpha-amylase.

2011-09-18T10:32:31Z

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme. To change the pKa of the catalytic residues in t...

J E Nielsen, L Beier, D Otzen, T V Borchert, H B Frantzen, K V Andersen, A Svendsen (1999) Eur J Biochem 264: 3 816-824

A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant.

2011-09-18T10:32:31Z

Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 unfolds...

D E Otzen, L Christiansen, M Schülein (1999) Protein Sci 8: 9 1878-1887

Salt-induced detour through compact regions of the protein folding landscape.

2011-09-18T10:32:31Z

In several cases, inorganic salts have been used to induce partly structured states in protein folding. But what is the nature of these states: Do they represent key stepping stones in the folding process, or are they circumstantial pitfalls in the energy landscape? Here we report that, in the case of the two-state protein S6, the salt-induced collapsed state is off the usual folding routes in the...

D E Otzen, M Oliveberg (1999) Proc Natl Acad Sci U S A 96: 21 11746-11751

Folding of circular and permuted chymotrypsin inhibitor 2: retention of the folding nucleus.

2011-09-18T10:32:31Z

The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nucleation-condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be al...

D E Otzen, A R Fersht (1998) Biochemistry 37: 22 8139-8146

The rate of isomerisation of peptidyl-proline bonds as a probe for interactions in the physiological denatured state of chymotrypsin inhibitor 2.

2011-09-18T10:32:31Z

There are four peptidyl-proline bonds in the 64-residue protein chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which...

Y J Tan, M Oliveberg, D E Otzen, A R Fersht (1997) J Mol Biol 269: 4 611-622

Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis.

2011-09-18T10:32:31Z

Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global ...

J L Neira, L S Itzhaki, D E Otzen, B Davis, A R Fersht (1997) J Mol Biol 270: 1 99-110

[Treatment of narcotic addiction--"fix" room causes less harm]. Interview by Jesper Berg].

2011-09-18T10:32:31Z

D Otzen, S Riegels (1997) Sygeplejersken 97: 46 14-15

Structure of the transition state for folding of a protein derived from experiment and simulation.

2011-09-18T10:32:31Z

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are ...

V Daggett, A Li, L S Itzhaki, D E Otzen, A R Fersht (1996) J Mol Biol 257: 2 430-440

Structural factors contributing to the hydrophobic effect: the partly exposed hydrophobic minicore in chymotrypsin inhibitor 2.

2011-09-18T10:32:31Z

The structural basis for the stability of a partly solvent-exposed hydrophobic minicore, formed by the residues Leu51, Val57, and Phe69 in the reactive loop of the serine protease inhibitor chymotrypsin inhibitor 2 (CI2), was analyzed from the stability of 17 mutant proteins, in which side chain methylene groups were deleted or rearranged. The mutations destabilize the protein by 0.3-4.8 kcal/mol,...

D E Otzen, M Rheinnecker, A R Fersht (1995) Biochemistry 34: 40 13051-13058

Protein fragments as models for events in protein folding pathways: protein engineering analysis of the association of two complementary fragments of the barley chymotrypsin inhibitor 2 (CI-2).

2011-09-18T10:32:31Z

Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of association of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies o...

J Ruiz-Sanz, G de Prat Gay, D E Otzen, A R Fersht (1995) Biochemistry 34: 5 1695-1701

Side-chain determinants of beta-sheet stability.

2011-09-18T10:32:31Z

beta-Sheet propensities of different amino acids depend on the context of both secondary and tertiary structure. In an attempt to establish general empirical relationships that determine this context dependence, we have determined the free energy of unfolding of a series of mutants at six positions in the beta-sheet of chymotrypsin inhibitor 2 (CI2). We have generated the series Val-->Ala-->Gly an...

D E Otzen, A R Fersht (1995) Biochemistry 34: 17 5718-5724

Movement of the position of the transition state in protein folding.

2011-09-18T10:32:31Z

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state an...

A Matouschek, D E Otzen, L S Itzhaki, S E Jackson, A R Fersht (1995) Biochemistry 34: 41 13656-13662

The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding.

2011-09-18T10:32:31Z

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 s...

L S Itzhaki, D E Otzen, A R Fersht (1995) J Mol Biol 254: 2 260-288

Nature and consequences of GroEL-protein interactions.

2011-09-18T10:32:31Z

The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL. The chaperonin retards the rate of refolding of wild type and 32 representative point mutants. The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on t...

L S Itzhaki, D E Otzen, A R Fersht (1995) Biochemistry 34: 44 14581-14587

Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway.

2011-09-18T10:32:31Z

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (C12) in order to study the development of structure on elongation of the polypeptide in solution. We present an extensive biophysical characterization of ten fragments using different conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (...

G de Prat Gay, J Ruiz-Sanz, J L Neira, F J Corrales, D E Otzen, A G Ladurner, A R Fersht (1995) J Mol Biol 254: 5 968-979

Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

2011-09-18T10:32:31Z

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of sec...

D E Otzen, L S Itzhaki, N F elMasry, S E Jackson, A R Fersht (1994) Proc Natl Acad Sci U S A 91: 22 10422-10425

Dual hydrolytic role for Pb(II) ions.

2011-09-18T10:32:31Z

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds ...

D E Otzen, J Barciszewski, B F Clark (1994) Biochimie 76: 1 15-21

Single versus parallel pathways of protein folding and fractional formation of structure in the transition state.

2011-09-18T10:32:31Z

Protein engineering and kinetic experiments indicate that some regions of proteins have partially formed structure in the transition state for protein folding. A crucial question is whether there is a genuine single transition state that has interactions that are weakened in those regions or there are parallel pathways involving many transition states, some with the interactions fully formed and o...

A R Fersht, L S Itzhaki, N F elMasry, J M Matthews, D E Otzen (1994) Proc Natl Acad Sci U S A 91: 22 10426-10429

Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture.

2011-09-18T10:32:31Z

DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold high...

J Barciszewski, S I Rattan, G E Siboska, D E Otzen, B F Clark (1993) FEBS Lett 318: 2 186-188

Altered lead(II)-cleavage pattern of free Phe-tRNAPhe and Phe-tRNAPhe in ternary complex with EF-Tu:GTP.

2011-09-18T10:32:31Z

Pb2+ ions in sub-millimolar concentrations are known to cleave internucleotide bonds of phenylalanine-specific transfer RNA (tRNAPhe) from Saccharomyces. cerevisiae specifically between nucleotides D17 and G18 in the D-loop, with additional minor cleavages after D16 and G15. This makes lead(II) a sensitive structural probe for correct folding of tRNAPhe. In the present paper we use Pb2+ ions as a ...

D E Otzen, J Barciszewski, B F Clark (1993) Biochem Mol Biol Int 31: 1 95-103

Specific incorporation of kinetin into eukaryotic and prokaryotic transfer ribonucleic acid molecules.

2011-09-18T10:32:31Z

We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase. We also show that kinetin is specifically incorporated into E. coli tRNA(Tyr) and most probably at position 37. To our knowledge, this is the first report of a nucleic acid base exchange rea...

J Barciszewski, D Otzen, S I Rattan, B F Clark (1992) Biochem Int 28: 5 805-811