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Daniele Dell'Orco

Daniele Dell'Orco, Ph.D.
Assistant Professor
Department of Life Sciences and Reproduction,
Section of Biological Chemistry, University of Verona,
strada le Grazie 8, 37134 Verona
Welcome to my publication list page! Here is a brief summary of my scientific activity and interests.

Short Biography:
I studied Physics and Biophysics at the University of Parma (Italy) and Lund (Sweden). I obtained a PhD in Biotechnology and Molecular Medicine from the University of Modena and Reggio Emilia (Italy). During my pre- and post-doctoral activity I visited several laboratories in Sweden and in Germany, to improve my knowledge of computational and experimental biophysical chemistry and systems biology. I have been A. von Humboldt Research Fellow at the University of Oldenburg (Germany) for almost 2.5 years. Since December 2011 I am Assistant Professor at the University of Verona, at the Biological Chemistry section of the Medical Faculty. On a regular basis I act as a referee for several peer-review journals in the fields of Biochemistry, Biophysical Chemistry, Systems Biology and biology-oriented nanoscience, among which:

- Molecular Biosystems, Journal of Physical Chemistry B, PhysChemChemPhys, BBA-Proteins and Proteomics, PLoS Computational Biology, Integrative Biology, Cellular and Molecular Life Sciences, BioMaterials, ACS Nano, PLoS One, BioSystems

I am an Editorial Board member for the open-access Journal of Nanomedicine and Biotherapeutic Discovery , as well as a member of the Biochemical Society (UK), the Italian society of Biochemistry and Molecular Biology (SIB) and the Bioinformatics Italian Society (BITS).

Research interests:
My main scientific activity is focused on the experimental and theoretical characterization of protein-protein and protein-ion interactions involved in vertebrate vision. This includes systems biology approaches to unravel the biochemical mechanisms behind complex cell behaviors in photoreceptors during normal and disease-associated conditions. I use a bunch of experimental biophysical techniques such as Surface Plasmon Resonance, Isothermal Titration Calorimetry, Circular Dichroism, Fluorescence and Absorption spectroscopies, in order to characterize the (thermo)dynamics and the kinetics of the processes under investigation. This allows me to obtain kinetic and thermodynamic constants that I then employ in mathematical models. I am also active in computational structural biology especially in the fast semi-empirical characterization of protein-protein interactions in normal and in disease-associated conditions by means of molecular simulation and docking algorithms, and bioinformatics techniques.
I am also involved in the analysis of nanoparticle-protein interactions in several biological fluids and the perturbations exerted by nanoparticle-protein complexes on biochemical processes.
My research also includes the biochemical and biophysical characterization of neuronal calcium sensor proteins, especially focused on the dynamics of conformational changes in response to Ca2+-binding.

Journal articles

Karl-Wilhelm Koch, Daniele Dell'Orco (2013)  A Calcium-Relay Mechanism in Vertebrate Phototransduction.   ACS chemical neuroscience 4: 6. 909–917 Mar  
Abstract: Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving ion channels, exchangers, and ion-pumps but also calcium-sensor proteins and their targets. Thus, control of physiological responses very often depends on incremental changes of the cytoplasmic calcium concentration, which are sensed by calcium-binding proteins and are further transmitted to specific target proteins. This Review will focus on calcium-signaling in vertebrate photoreceptor cells, where recent physiological and biochemical data indicate that a subset of neuronal calcium sensor proteins named guanylate cyclase-activating proteins (GCAPs) operate in a calcium-relay system, namely, to make gradual responses to small changes in calcium. We will further integrate this mechanism in an existing computational model of phototransduction showing that it is consistent and compatible with the dynamics that are characteristic for the precise operation of the phototransduction pathways.
Patrick Zägel, Daniele Dell'Orco, Karl-Wilhelm Koch (2013)  The Dimerization Domain in Outer Segment Guanylate Cyclase Is a Ca(2+)-Sensitive Control Switch Module.   Biochemistry in press: Jul  
Abstract: Membrane-bound guanylate cyclases harbor a region called the dimerization or linker domain, which aids the enzymes in adopting an optimal monomer-monomer arrangement for catalysis. One subgroup of these guanylate cyclases is expressed in rod and cone cells of vertebrate retina, and mutations in the dimerization domain of rod outer segment guanylate cyclase 1 (ROS-GC1, encoded by the GUCY2D gene) correlate with retinal cone-rod dystrophies. We investigate how a Q847L/K848Q double mutation, which was found in patients suffering from cone-rod dystrophy, and the Q847L and K848Q single-point mutations affect the regulatory mechanism of ROS-GC1. Both the wild type and mutants of heterologously expressed ROS-GC1 were present in membranes. However, the mutations affected the catalytic properties of ROS-GC1 in different manners. All mutants had higher basal guanylate cyclase activities but lower levels of activation by Ca(2+)-sensing guanylate cyclase-activating proteins (GCAPs). Further, incubation with wild-type GCAP1 and GCAP2 revealed for all ROS-GC1 mutants a shift in Ca(2+) sensitivity, but activation of the K848Q mutant by GCAPs was severely impaired. Apparent affinities for GCAP1 and GCAP2 were different for the double mutant and the wild type. Circular dichroism spectra of the dimerization domain showed that the wild type and mutants adopt a prevalently α-helical structure, but mutants exhibited lower thermal stability. Our results indicate that the dimerization domain serves as a Ca(2+)-sensitive control module. Although it is per se not a Ca(2+)-sensing unit, it seems to integrate and process information regarding Ca(2+) sensing by sensor proteins and regulator effector affinity.
Brandon M Invergo, Ludovica Montanucci, Karl-Wilhelm Koch, Jaume Bertranpetit, Daniele Dell'Orco (2013)  Exploring the rate-limiting steps in visual phototransduction recovery by bottom-up kinetic modeling.   Cell communication and signaling : CCS 11: 1. 05  
Abstract: BACKGROUND: Phototransduction in vertebrate photoreceptor cells represents a paradigm of signaling pathways mediated by G-protein-coupled receptors (GPCRs), which share common modules linking the initiation of the cascade to the final response of the cell. In this work, we focused on the recovery phase of the visual photoresponse, which is comprised of several interacting mechanisms. RESULTS: We employed current biochemical knowledge to investigate the response mechanisms of a comprehensive model of the visual phototransduction pathway. In particular, we have improved the model by implementing a more detailed representation of the recoverin (Rec)-mediated calcium feedback on rhodopsin kinase and including a dynamic arrestin (Arr) oligomerization mechanism. The model was successfully employed to investigate the rate limiting steps in the recovery of the rod photoreceptor cell after illumination. Simulation of experimental conditions in which the expression levels of rhodospin kinase (RK), of the regulator of the G-protein signaling (RGS), of Arr and of Rec were altered individually or in combination revealed severe kinetic constraints to the dynamics of the overall network. CONCLUSIONS: Our simulations confirm that RGS-mediated effector shutdown is the rate-limiting step in the recovery of the photoreceptor and show that the dynamic formation and dissociation of Arr homodimers and homotetramers at different light intensities significantly affect the timing of rhodopsin shutdown. The transition of Arr from its oligomeric storage forms to its monomeric form serves to temper its availability in the functional state. Our results may explain the puzzling evidence that overexpressing RK does not influence the saturation time of rod cells at bright light stimuli. The approach presented here could be extended to the study of other GPCR signaling pathways.
Marco Aquila, Mascia Benedusi, Karl-Wilhelm Koch, Daniele Dell'Orco, Giorgio Rispoli (2013)  Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin.   Cell Calcium 53: 3. 180-186 Mar  
Abstract: The Ca(2+) modulation of pore formation (and disaggregation) kinetics of a synthetic analog of alamethicin F50/5 ([l-Glu(OMe)(7,18,19)]), a potent antibiotic peptide, was investigated in situ and in vitro. The in situ experiments consisted in whole-cell recording from isolated retinal rod outer segments (OS), because once blocking the only OS endogenous conductance with saturating light, the current flows entirely through the (exogenous) channels formed by the peptide. The kinetics of current change induced by peptide application and removal (in â¼50ms) on the OS extracellular side was measured in the presence of divalent cations at different concentrations. The in vitro experiments consisted on the divalent cations modulation of [l-Glu(OMe)(7,18,19)] binding to a mimetic OS membrane immobilized on a sensor chip surface, employing surface plasmon resonance spectroscopy (SPR). The presence of even low mM Ca(2+) or Mg(2+) sufficed to increase the [l-Glu(OMe)(7,18,19)] apparent affinity for the mimetic OS membrane up to â¼4-fold, which accelerated the activation of the peptide-induced current in OS by â¼10-fold with respect to low Ca(2+). In situ and in vitro experiments indicate that high concentrations of divalent cations increased also membrane rigidity, contrasting their effect on increasing the pore formation rate.
Lorenzo Cangiano, Daniele Dell'Orco (2013)  Detecting single photons: A supramolecular matter?   FEBS Lett 587: 1. 1-4 Jan  
Abstract: Rod photoreceptors detect single photons through a tradeoff of light collecting ability, amplification and speed. Key roles are played by rhodopsin (Rh) and transducin (Gt), whose complex supramolecular organization in outer segment disks begs for a functional interpretation. Here we review past and recent evidence of a temperature-dependence of photon detection by mammalian rods, and link this phenomenon with the putative oligomeric organization of Rh and new ideas on the dynamics of Rh-Gt interaction. Identifying an electrophysiological correlate of the supramolecular organization of Rh and Gt may shed light on the evolutionary advantage it confers to night vision.
Daniele Dell'Orco (2013)  A physiological role for the supramolecular organization of rhodopsin and transducin in rod photoreceptors.   FEBS letters 587: 13. 2060-2066 Jun  
Abstract: Vertebrate vision in rod photoreceptors begins when a photon hits the visual pigment rhodopsin (Rh) and triggers the phototransduction cascade. Although the fine biochemical and biophysical details of this paradigmatic signalling pathway have been studied for decades, phototransduction still presents unclear mechanistic aspects. Increasing lines of evidence suggest that the visual pigment rhodopsin (Rh) is natively organized in dimers on the surface of disc membranes, and may form higher order "paracrystalline" assemblies, which are not easy to reconcile with the classical collision-coupling mechanistic scenario evoked to explain the extremely fast molecular processes required in phototransduction. The questioned and criticized existence of paracrystalline Rh rafts can be fully accepted only if it can be explained in functional terms by a solid mechanistic picture. Here we discuss how recent data suggest a physiological role for supramolecular assemblies of Rh and its cognate G protein transducin (Gt), which by forming transient complexes in the dark may ensure rapid activation of the cascade even in a crowded environment that, according to the classical picture, would otherwise stop the cascade.
Daniele Dell'Orco, Stefan Sulmann, Sara Linse, Karl-Wilhelm Koch (2012)  Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device.   Anal Chem 84: 6. 2982-2989 Mar  
Abstract: Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.
Daniele Dell'orco, Martin Lundqvist, Tommy Cedervall, Sara Linse (2012)  Delivery success rate of engineered nanoparticles in the presence of the protein corona: a systems-level screening.   Nanomedicine Feb  
Abstract: Nanoparticles (NPs) for medical applications are often introduced into the body via intravenous injections, leading to the formation of a protein corona on their surface due to the interaction with blood plasma proteins. Depending on its composition and time evolution, the corona will modify the biological behavior of the particle. For successful delivery and targeting, it is therefore important to assess on a quantitative basis how and to what extent the presence of the corona perturbs the specific interaction of a designed NP with its cellular target. We present a theoretical systems-level analysis, in which peptides have been covalently coupled to the surface of nanoparticles, describing the delivery success rate in varying conditions, with regard to protein composition of the surrounding fluid. Dynamic modeling and parameter sensitivity analysis proved to be useful and computationally affordable tools to aid in the design of NPs with increased success rate probability in a biological context.
Daniele Dell'Orco, Martin Lundqvist, Tommy Cedervall, Sara Linse (2012)  Delivery success rate of engineered nanoparticles in the presence of the protein corona: a systems-level screening.   Nanomedicine 8: 8. 1271-1281 Nov  
Abstract: Nanoparticles (NPs) for medical applications are often introduced into the body via intravenous injections, leading to the formation of a protein corona on their surface due to the interaction with blood plasma proteins. Depending on its composition and time evolution, the corona will modify the biological behavior of the particle. For successful delivery and targeting, it is therefore important to assess on a quantitative basis how and to what extent the presence of the corona perturbs the specific interaction of a designed NP with its cellular target. We present a theoretical systems-level analysis, in which peptides have been covalently coupled to the surface of nanoparticles, describing the delivery success rate in varying conditions, with regard to protein composition of the surrounding fluid. Dynamic modeling and parameter sensitivity analysis proved to be useful and computationally affordable tools to aid in the design of NPs with increased success rate probability in a biological context. FROM THE CLINICAL EDITOR: The formation of a protein corona consisting of blood plasma proteins on the surface of intravenously delivered nanoparticles may modify the biological behavior of the particles. This team of investigators present a theoretical systems-level analysis of this important and often neglected phenomenon.
Evgeni Yu Zernii, Konstantin E Komolov, Sergei E Permyakov, Tatiana Kolpakova, Daniele Dell'Orco, Annika Poetzsch, Ekaterina L Knyazeva, Ilya I Grigoriev, Eugene A Permyakov, Ivan I Senin, Pavel P Philippov, Karl-Wilhelm Koch (2011)  Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase.   Biochem J 435: 2. 441-450 Apr  
Abstract: NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-Ï interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.
Daniele Dell'Orco, Karl-Wilhelm Koch (2011)  A dynamic scaffolding mechanism for rhodopsin and transducin interaction in vertebrate vision.   Biochem J 440: 2. 263-271 Dec  
Abstract: The early steps in vertebrate vision require fast interactions between Rh (rhodopsin) and Gt (transducin), which are classically described by a collisional coupling mechanism driven by the free diffusion of monomeric proteins on the disc membranes of rod and cone cells. Recent findings, however, point to a very low mobility for Rh and support a substantially different supramolecular organization. Moreover, Rh-Gt interactions seem to possibly occur even prior to light stimuli, which is also difficult to reconcile with the classical scenario. We investigated the kinetics of interaction between native Rh and Gt in different conditions by surface plasmon resonance and analysed the results in the general physiological context by employing a holistic systems modelling approach. The results from the present study point to a mechanism that is intermediate between pure collisional coupling and physical scaffolding. Such a 'dynamic scaffolding', in which prevalently dimeric Rh and Gt interact in the dark by forming transient complexes (~25% of Gt is precoupled to Rh), does not slow down the phototransduction cascade, but is compatible with the observed photoresponses on a broad scale of light stimuli. We conclude that Rh molecules and Rh-Gt complexes can both absorb photons and trigger the visual cascade.
Daniele Dell'Orco, Martin Lundqvist, Cecilia Oslakovic, Tommy Cedervall, Sara Linse (2010)  Modeling the time evolution of the nanoparticle-protein corona in a body fluid.   PLoS One 5: 6. 06  
Abstract: BACKGROUND: Nanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona. RESULTS: We developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL. CONCLUSIONS: The simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine.
Daniele Dell'Orco, Karl-Wilhelm Koch (2010)  Systems biochemistry approaches to vertebrate phototransduction: towards a molecular understanding of disease.   Biochem Soc Trans 38: 5. 1275-1280 Oct  
Abstract: Phototransduction in vertebrates represents a paradigm of signalling pathways, in particular those mediated by G-protein-coupled receptors. The variety of protein-protein, protein-ion and protein-nucleotide interactions makes up an intricate network which is finely regulated by activating-deactivating molecules and chemical modifications. The holistic systems properties of the network allow for typical adaptation mechanisms, which ultimately result in fine adjustments of sensitivity and electrical response of the photoreceptor cells to the broad range of light stimuli. In the present article, we discuss a novel bottom-up strategy to study the phototransduction cascade in rod cells starting from the underlying biochemistry. The resulting network model can be simulated and the predicted dynamic behaviour directly compared with data from electrophysiological experiments performed on a wide range of illumination conditions. The advantage of applying procedures typical of systems theory to a well-studied signalling pathway is also discussed. Finally, the potential application to the study of the molecular basis of retinal diseases is highlighted through a practical example, namely the simulation of conditions related to Leber congenital amaurosis.
Petra Behnen, Daniele Dell'Orco, Karl-Wilhelm Koch (2010)  Involvement of the calcium sensor GCAP1 in hereditary cone dystrophies.   Biol Chem 391: 6. 631-637 Jun  
Abstract: Progressive visual impairment leading to blindness is often associated with inherited retinal dystrophies. These disorders correlate in most cases with mutations in genes that code for proteins of the visual transduction system in rod and cone photoreceptor cells. Recent progress has highlighted the involvement of a neuronal calcium sensor protein that is specifically expressed in rod and cone cells and operates as a guanylate cyclase-activating protein (GCAP). A group of patients suffering from cone or cone-rod dystrophies carry mutations in the GCAP1 gene, and biochemical analysis of GCAP1 function revealed that for most of these mutations GCAP1 exhibits a disturbance in its Ca(2+)-sensing and its guanylate cyclase-activating properties. Cellular consequences of different GCAP1 mutations are compared and discussed.
Daniele Dell'Orco, Petra Behnen, Sara Linse, Karl-Wilhelm Koch (2010)  Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy.   Cell Mol Life Sci 67: 6. 973-984 Mar  
Abstract: Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca(2+) sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca(2+)-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca(2+) binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca(2+) affinity and thermal stability, whereas all the mutants showed decreased Ca(2+) affinity and significantly lower thermal stability in both apo and Ca(2+)-loaded forms. No apparent cooperativity of Ca(2+) binding was detected for any variant. Finally, the non-myristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca(2+) concentrations. Thus, we conclude that distorted Ca(2+)-sensor properties could lead to cone dysfunction.
Daniele Dell'Orco, Michael Müller, Karl-Wilhelm Koch (2010)  Quantitative detection of conformational transitions in a calcium sensor protein by surface plasmon resonance.   Chem Commun (Camb) Sep  
Abstract: We determined the conditions under which surface plasmon resonance can be used to monitor at real-time the Ca(2+)-induced conformational transitions of the sensor protein recoverin immobilized over a sensor chip. The equilibrium and the kinetics of conformational transitions were detected and quantified over a physiological range of Ca(2+) and protein concentrations similar to those found within cells. Structural analysis suggests that the detection principle reflects changes in the hydrodynamic properties of the protein and is not due to a mass effect. The phenomenon appears to be related to changes in the refractive index at the metal/dielectric interface.
Daniele Dell'Orco, Henning Schmidt, Simona Mariani, Francesca Fanelli (2009)  Network-level analysis of light adaptation in rod cells under normal and altered conditions.   Mol Biosyst 5: 10. 1232-1246 Oct  
Abstract: Photoreceptor cells finely adjust their sensitivity and electrical response according to changes in light stimuli as a direct consequence of the feedback and regulation mechanisms in the phototransduction cascade. In this study, we employed a systems biology approach to develop a dynamic model of vertebrate rod phototransduction that accounts for the details of the underlying biochemistry. Following a bottom-up strategy, we first reproduced the results of a robust model developed by Hamer et al. (Vis. Neurosci., 2005, 22(4), 417), and then added a number of additional cascade reactions including: (a) explicit reactions to simulate the interaction between the activated effector and the regulator of G-protein signalling (RGS); (b) a reaction for the reformation of the G-protein from separate subunits; (c) a reaction for rhodopsin (R) reconstitution from the association of the opsin apoprotein with the 11-cis-retinal chromophore; (d) reactions for the slow activation of the cascade by opsin. The extended network structure successfully reproduced a number of experimental conditions that were inaccessible to prior models. With a single set of parameters the model was able to predict qualitative and quantitative features of rod photoresponses to light stimuli ranging over five orders of magnitude, in normal and altered conditions, including genetic manipulations of the cascade components. In particular, the model reproduced the salient dynamic features of the rod from Rpe65(-/-) animals, a well established model for Leber congenital amaurosis and vitamin A deficiency. The results of this study suggest that a systems-level approach can help to unravel the adaptation mechanisms in normal and in disease-associated conditions on a molecular basis.
Daniele Dell'Orco (2009)  Fast predictions of thermodynamics and kinetics of protein-protein recognition from structures: from molecular design to systems biology.   Mol Biosyst 5: 4. 323-334 Apr  
Abstract: The increasing call for an overall picture of the interactions between the components of a biological system that give rise to the observed function is often summarized by the expression systems biology. Both the interpretative and predictive capabilities of holistic models of biochemical systems, however, depend to a large extent on the level of physico-chemical knowledge of the individual molecular interactions making up the network. This review is focused on the structure-based quantitative characterization of protein-protein interactions, ubiquitous in any biochemical pathway. Recently developed, fast and effective computational methods are reviewed, which allow the assessment of kinetic and thermodynamic features of the association-dissociation processes of protein complexes, both in water soluble and membrane environments. The performance and the accuracy of fast and semi-empirical structure-based methods have reached comparable levels with respect to the classical and more elegant molecular simulations. Nevertheless, the broad accessibility and lower computational cost provide the former methods with the advantageous possibility to perform systems-level analyses including extensive in silico mutagenesis screenings and large-scale structural predictions of multiprotein complexes.
Francesca Fanelli, Daniele Dell'orco (2008)  Dark and photoactivated rhodopsin share common binding modes to transducin.   FEBS Lett 582: 6. 991-996 Mar  
Abstract: The structure of the photoactivated deprotonated rhodopsin intermediate was compared with two different structures of dark rhodopsin. Structure comparisons relied on the computation of molecular indices and on docking simulations with heterotrimeric transducin (Gt). The results of this study provide the first evidence that dark and photoactivated rhodopsins share a common recognition mode to Gt, characterized by the docking of the Gt alpha C-tail in the proximity to the E/DRY motif of rhodopsin.
Daniele Dell'Orco, Henning Schmidt (2008)  Mesoscopic Monte Carlo simulations of stochastic encounters between photoactivated rhodopsin and transducin in disc membranes.   J Phys Chem B 112: 14. 4419-4426 Apr  
Abstract: The issue of how the molecular organization of rod outer segments (ROS) discs affects the initial timing of the photoresponse in vertebrates has been recently raised by novel structural findings that raise doubts about the classical scenario of monomeric rhodopsin (R) and heterotrimeric transducin (G) freely diffusing in the membrane milieu. In this study, we investigate this issue by means of mesoscopic Monte Carlo (MMC) simulations of the stochastic encounters between one photoactivated R and one G, explicitly taking into account the molecular size and the diffusion coefficient of each species as well as crowding effects. Three different scenarios were compared with respect to their effects on timing, namely, (a) the classical framework, where both G and monomeric R are allowed to freely diffuse in the ROS disc membrane, (b) the ideal paracrystalline organization of R dimers considered as a structural unit, where ordered rows completely cover the disc membrane patch, and (c) the scenario suggested by recent AFM data, where R dimers organize in differently sized rafts with varying local concentrations. Our simulations suggest that a similar kinetic response could arise from very different microscopic scenarios, thus opening new interpretations to the controversial recent findings. Moreover, we show that if high-density R packing on ROS discs is characterized by a highly ordered structural organization rather than unspecific aggregation, an unexpected favorable effect on the temporal response of early phototransduction reactions can occur.
Daniele Dell'Orco, Daniele Casciari, Francesca Fanelli (2008)  Quaternary structure predictions and estimation of mutational effects on the free energy of dimerization of the OMPLA protein.   J Struct Biol 163: 2. 155-162 Aug  
Abstract: This study represents an extension to the outer membrane phospholipase A protein (OMPLA) of the docking-based protocols previously developed for quaternary structure predictions of transmembrane oligomeric proteins and for estimating mutational effects on the thermodynamics of protein-protein and protein-DNA association. Predictions of the likely architecture of OMPLA homo-dimers were carried out on 31 different forms of the monomer, 30 of which were variants of the unbound state. In all the test cases but the ones characterized by combined deletions of the 98-110 and 145-153 segments (L2 and L3, respectively), native-like complexes could be predicted, independent of the bound or unbound state of the structural model, of side chain conformation and presence or absence of amino acid deletions at the putative inter-monomer interface. The protocol for estimating mutational effects on the thermodynamics of protein-protein association proved effective as well. In fact, it was possible to estimate correctly the effects of five mutants on the free energy of dimerization of the sulfonylated form of OMPLA. The integrity of L2 and either one of the L1, L3 and L4 loops turned out to be more important than sulfonylation for the achievement of the native dimeric architecture. On the other hand, sulfonylation seems to be essential for a favorable dimerization energetics.
Daniele Casciari, Daniele Dell'Orco, Francesca Fanelli (2008)  Homodimerization of neurotensin 1 receptor involves helices 1, 2, and 4: insights from quaternary structure predictions and dimerization free energy estimations.   J Chem Inf Model 48: 8. 1669-1678 Aug  
Abstract: A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been combined with a protocol for dimerization free energy estimations to predict likely interfaces in the neurotensin 1 receptor (NTS1) homodimers. The results of this study suggest that the likely intermonomer interfaces compatible with in vitro binding affinity constants essentially involve helices 1, 2, and 4 and do not include disulfide bridges. The correlative model initially developed on Glycophorin A and herein extended to a G protein-Coupled Receptor (GPCR) appears to be a useful tool for estimating the association free energies of transmembrane proteins independent of the size and shape of the interface. In the desirable future cases, in which in vitro intermonomer binding affinities will be available for other GPCRs, such a correlative model will work as an additional criterion for helping in the selection of the most likely dimers.
Daniele Dell'orco, Pier Giuseppe De Benedetti (2008)  Quantitative structure-activity relationship analysis of canonical inhibitors of serine proteases.   J Comput Aided Mol Des 22: 6-7. 469-478 Jun/Jul  
Abstract: Correlation analysis was carried out between binding affinity data values from the literature and physicochemical molecular descriptors of two series of single point mutated canonical inhibitors of serine proteases, namely bovine pancreatic trypsin inhibitor (BPTI) and turkey ovomucoid third domain (OMTKY3), toward seven enzymes. Simple quantitative structure-activity relationship (QSAR) models based on either single or double linear regressions (SLR or DLR) were obtained, which highlight the role of hydrophobic and bulk/polarizability features of mutated amino acids of the inhibitors in modulating both affinity and specificity. The utility of the QSAR paradigm applied to the analysis of mutagenesis data was underlined, resulting in a simple tool to quantitatively help deciphering structure-function/activity relationships (SFAR) of different protein systems.
Daniele Dell'Orco, Michele Seeber, Francesca Fanelli (2007)  Monomeric dark rhodopsin holds the molecular determinants for transducin recognition: insights from computational analysis.   FEBS Lett 581: 5. 944-948 Mar  
Abstract: In this computational study, we have investigated the implications of rhodopsin (Rho) oligomerization in transducin (Gt) recognition. The results of docking simulations between heterotrimeric Gt and monomeric, dimeric and tetrameric inactive Rho corroborate the hypothesis that Rho and Gt can be found coupled already in the dark. Moreover, our extensive computational analysis suggests that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are contained in one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit.
Daniele Dell'Orco, Pier Giuseppe De Benedetti, Francesca Fanelli (2007)  In silico screening of mutational effects on transmembrane helix dimerization: insights from rigid-body docking and molecular dynamics simulations.   J Phys Chem B 111: 30. 9114-9124 Aug  
Abstract: In this study, a docking-based protocol has been probed for its ability to predict the effects of 32 single and double mutations on glycophorin A (GpA) homodimerization. Rigid-body docking simulations have been paralleled by molecular dynamics (MD) simulations in implicit membrane. The rigid-body docking-based approach proved effective in reconstituting the native architecture of the GpA dimer for the wild type and the wild-type-like mutants. The good correlative models between the intermolecular interaction descriptors derived both by rigid-body docking simulations and by MD simulations and experimental relative free energies support the assumption that the mutation-induced changes in the association free energy of GpA helices are essentially ascribed to differences in the packing interactions, whereas almost all the variations in the entropic contributions to the association free energy are constant and/or negligible. The MD-based models achieved provide insights into the structural determinants for disruptive and restoring mutational effects. The computational approaches presented in this study are fast and effective, and constitute simple and promising tools for in silico screening of mutational effects on the association properties of integral membrane proteins.
Daniele Dell'Orco, Pier Giuseppe De Benedetti, Francesca Fanelli (2007)  In silico screening of mutational effects on enzyme-proteic inhibitor affinity: a docking-based approach.   BMC Struct Biol 7: 06  
Abstract: BACKGROUND: Molecular recognition between enzymes and proteic inhibitors is crucial for normal functioning of many biological pathways. Mutations in either the enzyme or the inhibitor protein often lead to a modulation of the binding affinity with no major alterations in the 3D structure of the complex. RESULTS: In this study, a rigid body docking-based approach has been successfully probed in its ability to predict the effects of single and multiple point mutations on the binding energetics in three enzyme-proteic inhibitor systems. The only requirement of the approach is an accurate structural model of the complex between the wild type forms of the interacting proteins, with the assumption that the architecture of the mutated complexes is almost the same as that of the wild type and no major conformational changes occur upon binding. The method was applied to 23 variants of the ribonuclease inhibitor-angiogenin complex, to 15 variants of the barnase-barstar complex, and to 8 variants of the bovine pancreatic trypsin inhibitor-beta Trypsin system, leading to thermodynamic and kinetic estimates consistent with in vitro data. Furthermore, simulations with and without explicit water molecules at the protein-protein interface suggested that they should be included in the simulations only when their positions are well defined both in the wild type and in the mutants and they result to be relevant for the modulation of mutational effects on the association process. CONCLUSION: The correlative models built in this study allow for predictions of mutational effects on the thermodynamics and kinetics of association of three substantially different systems, and represent important extensions of our computational approach to cases in which it is not possible to estimate the absolute free energies. Moreover, this study is the first example in the literature of an extensive evaluation of the correlative weights of the single components of the ZDOCK score on the thermodynamics and kinetics of binding of protein mutants compared to the native state.Finally, the results of this study corroborate and extend a previously developed quantitative model for in silico predictions of absolute protein-protein binding affinities spanning a wide range of values, i.e. from -10 up to -21 kcal/mol. The computational approach is simple and fast and can be used for structure-based design of protein-protein complexes and for in silico screening of mutational effects on protein-protein recognition.
Daniele Dell'Orco, Michele Seeber, Pier Giuseppe De Benedetti, Francesca Fanelli (2005)  Probing fragment complementation by rigid-body docking: in silico reconstitution of calbindin D9k.   J Chem Inf Model 45: 5. 1429-1438 Sep/Oct  
Abstract: Fragment complementation is gaining an increasing impact as a nonperturbing method to probe noncovalent interactions within protein supersecondary structures. In this study, the fast Fourier transform rigid-body docking algorithm ZDOCK has been employed for in silico reconstitution of the calcium binding protein calbindin D9k, from its two EF-hands subdomains, namely, EF1 (residues 1-43) and EF2 (residues 44-75). The EF1 fragment has been used both in its wild type and in nine mutant forms, in line with in vitro experiments. Consistent with in vitro data, ZDOCK reconstituted the proper fold of wild-type and mutated calbindin, locating the nativelike structures (i.e., holding a root-mean-square deviation < 1 A with respect to the X-ray structure) among the first 10 top-scored solutions out of 4000. Moreover, the three independent in silico reconstitutions of wild-type calbindin ranked a nativelike structure at the top of the output list, that is, the best scored one. The algorithm has been also successfully challenged in reconstituting the EF2 homodimer from two identical copies of the monomer. Furthermore, quantitative models consisting of linear correlations between thermodynamic data and ZDOCK scores were built, providing a tested tool for very fast in silico predictions of the free energy of association of protein-protein complexes solved at the atomic level and known to not undergo significant conformational changes upon binding.
Francesca Fanelli, Daniele Dell'Orco (2005)  Rhodopsin activation follows precoupling with transducin: inferences from computational analysis.   Biochemistry 44: 45. 14695-14700 Nov  
Abstract: The electrostatic and shape complementarities between the crystal structures of dark rhodopsin and heterotrimeric transducin (Gt) have been evaluated by exhaustively sampling the roto-translational space of one protein with respect to the other. Structural complementarity, reliability, and consistency with in vitro evidence all converge in the same rhodopsin-Gt complex, showing that the functionally important R135 of the E/DRY motif is almost accessible to the C-terminus of Gt(alpha) already in the dark state. The main inference from this study is that activation of rhodopsin and Gt may be concurrent processes, consisting of conformational changes in a supramolecular complex formed prior to the light-induced activation of the photoreceptor.
Daniele Dell'Orco, Wei-Feng Xue, Eva Thulin, Sara Linse (2005)  Electrostatic contributions to the kinetics and thermodynamics of protein assembly.   Biophys J 88: 3. 1991-2002 Mar  
Abstract: The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.
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