Abstract: The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).
Abstract: Previous experiments examining the transcriptional profile of the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of the Fur regulon in response to various environmental stressors. To test the involvement of Fur in the growth response and transcriptional regulation of D. vulgaris, a targeted mutagenesis procedure was used for deleting the fur gene. Growth of the resulting Deltafur mutant (JW707) was not affected by iron availability, but the mutant did exhibit increased sensitivity to nitrite and osmotic stresses compared to the wild type. Transcriptional profiling of JW707 indicated that iron-bound Fur acts as a traditional repressor for ferrous iron uptake genes (feoAB) and other genes containing a predicted Fur binding site within their promoter. Despite the apparent lack of siderophore biosynthesis genes within the D. vulgaris genome, a large 12-gene operon encoding orthologs to TonB and TolQR also appeared to be repressed by iron-bound Fur. While other genes predicted to be involved in iron homeostasis were unaffected by the presence or absence of Fur, alternative expression patterns that could be interpreted as repression or activation by iron-free Fur were observed. Both the physiological and transcriptional data implicate a global regulatory role for Fur in the sulfate-reducing bacterium D. vulgaris.
Abstract: Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >/= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-gel electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.
Abstract: The molecular response of Shewanella oneidensis MR-1 to variations in extracellular pH was investigated based on genomewide gene expression profiling. Microarray analysis revealed that cells elicited both general and specific transcriptome responses when challenged with environmental acid (pH 4) or base (pH 10) conditions over a 60-min period. Global responses included the differential expression of genes functionally linked to amino acid metabolism, transcriptional regulation and signal transduction, transport, cell membrane structure, and oxidative stress protection. Response to acid stress included the elevated expression of genes encoding glycogen biosynthetic enzymes, phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS), whereas the molecular response to alkaline pH was characterized by upregulation of nhaA and nhaR, which are predicted to encode an Na+/H+ antiporter and transcriptional activator, respectively, as well as sulfate transport and sulfur metabolism genes. Collectively, these results suggest that S. oneidensis modulates multiple transporters, cell envelope components, and pathways of amino acid consumption and central intermediary metabolism as part of its transcriptome response to changing external pH conditions.
Abstract: BACKGROUND: Differentially expressed genes are typically identified by analyzing the variation between replicate measurements. These procedures implicitly assume that there are no systematic errors in the data even though several sources of systematic error are known. RESULTS: OpWise estimates the amount of systematic error in bacterial microarray data by assuming that genes in the same operon have matching expression patterns. OpWise then performs a Bayesian analysis of a linear model to estimate significance. In simulations, OpWise corrects for systematic error and is robust to deviations from its assumptions. In several bacterial data sets, significant amounts of systematic error are present, and replicate-based approaches overstate the confidence of the changers dramatically, while OpWise does not. Finally, OpWise can identify additional changers by assigning genes higher confidence if they are consistent with other genes in the same operon. CONCLUSION: Although microarray data can contain large amounts of systematic error, operons provide an external standard and allow for reasonable estimates of significance. OpWise is available at http://microbesonline.org/OpWise.
Abstract: The physiology and transcriptome dynamics of the metal ion-reducing bacterium Shewanella oneidensis strain MR-1 in response to nonradioactive strontium (Sr) exposure were investigated. Studies indicated that MR-1 was able to grow aerobically in complex medium in the presence of 180 mM SrCl2 but showed severe growth inhibition at levels above that concentration. Temporal gene expression profiles were generated from aerobically grown, mid-exponential-phase MR-1 cells shocked with 180 mM SrCl2 and analyzed for significant differences in mRNA abundance with reference to data for nonstressed MR-1 cells. Genes with annotated functions in siderophore biosynthesis and iron transport were among the most highly induced (>100-fold [P < 0.05]) open reading frames in response to acute Sr stress, and a mutant (SO3032::pKNOCK) defective in siderophore production was found to be hypersensitive to SrCl2 exposure, compared to parental and wild-type strains. Transcripts encoding multidrug and heavy metal efflux pumps, proteins involved in osmotic adaptation, sulfate ABC transporters, and assimilative sulfur metabolism enzymes also were differentially expressed following Sr exposure but at levels that were several orders of magnitude lower than those for iron transport genes. Precipitate formation was observed during aerobic growth of MR-1 in broth cultures amended with 50, 100, or 150 mM SrCl2 but not in cultures of the SO3032::pKNOCK mutant or in the abiotic control. Chemical analysis of this precipitate using laser-induced breakdown spectroscopy and static secondary ion mass spectrometry indicated extracellular solid-phase sequestration of Sr, with at least a portion of the heavy metal associated with carbonate phases.
Abstract: The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.
Abstract: Two-component systems including histidine protein kinases represent the primary signal transduction paradigm in prokaryotic organisms. To understand how these systems adapt to allow organisms to detect niche-specific signals, we analyzed the phylogenetic distribution of nearly 5,000 histidine protein kinases from 207 sequenced prokaryotic genomes. We found that many genomes carry a large repertoire of recently evolved signaling genes, which may reflect selective pressure to adapt to new environmental conditions. Both lineage-specific gene family expansion and horizontal gene transfer play major roles in the introduction of new histidine kinases into genomes; however, there are differences in how these two evolutionary forces act. Genes imported via horizontal transfer are more likely to retain their original functionality as inferred from a similar complement of signaling domains, while gene family expansion accompanied by domain shuffling appears to be a major source of novel genetic diversity. Family expansion is the dominant source of new histidine kinase genes in the genomes most enriched in signaling proteins, and detailed analysis reveals that divergence in domain structure and changes in expression patterns are hallmarks of recent expansions. Finally, while these two modes of gene acquisition are widespread across bacterial taxa, there are clear species-specific preferences for which mode is used.
Abstract: Many of the proteins that are candidates for bioenergetic pathways involved with sulfate respiration in Desulfovibrio spp. have been studied, but complete pathways and overall cell physiology remain to be resolved for many environmentally relevant conditions. In order to understand the metabolism of these microorganisms under adverse environmental conditions for improved bioremediation efforts, Desulfovibrio vulgaris Hildenborough was used as a model organism to study stress response to nitrite, an important intermediate in the nitrogen cycle. Previous physiological studies demonstrated that growth was inhibited by nitrite and that nitrite reduction was observed to be the primary mechanism of detoxification. Global transcriptional profiling with whole-genome microarrays revealed coordinated cascades of responses to nitrite in pathways of energy metabolism, nitrogen metabolism, oxidative stress response, and iron homeostasis. In agreement with previous observations, nitrite-stressed cells showed a decrease in the expression of genes encoding sulfate reduction functions in addition to respiratory oxidative phosphorylation and ATP synthase activity. Consequently, the stressed cells had decreased expression of the genes encoding ATP-dependent amino acid transporters and proteins involved in translation. Other genes up-regulated in response to nitrite include the genes in the Fur regulon, which is suggested to be involved in iron homeostasis, and genes in the Per regulon, which is predicted to be responsible for oxidative stress response.
Abstract: Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.
Abstract: Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution may be driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysA and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although genes within operons are usually closely spaced because of a neutral bias toward deletion and because of selection against large overlaps, genes in highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution may be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.
Abstract: Whole-genomic expression patterns were examined in Shewanella oneidensis cells exposed to elevated sodium chloride. Genes involved in Na(+) extrusion and glutamate biosynthesis were significantly up-regulated, and the majority of chemotaxis/motility-related genes were significantly down-regulated. The data also suggested an important role for metabolic adjustment in salt stress adaptation in S. oneidensis.
Abstract: We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacter pylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, and its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from six phylogenetically diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC 6803 has many operons even though it has unusually wide spacings between conserved adjacent genes.
Abstract: The organization of bacterial genes into operons was originally ascribed to the benefits of co-regulation. More recently, the "selfish operon" model, in which operons are formed by repeated gain and loss of genes, was proposed. Indeed, operons are often subject to horizontal gene transfer (HGT). On the other hand, non-HGT genes are particularly likely to be in operons. To clarify whether HGT is involved in operon formation, we identified recently formed operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli--indicating HGT--form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent with the species tree. In contrast, essential genes and ubiquitous genes without paralogs--genes believed to undergo HGT rarely--often form new operons. We conclude that HGT is not a cause of operon formation but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns and infer that operons should be more likely to evolve than independent promoters when regulation is complex. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.
Abstract: In bacteria, most genes are on the leading strand of replication, a phenomenon attributed to collisions between the DNA and RNA polymerases. In Escherichia coli, these collisions slow the movement of the replication fork through actively transcribed genes only if they are coded on the lagging strand. For genes on both strands, however, these collisions sever nascent transcripts and interrupt gene expression. Based on these observations, we propose a new theory to explain strand bias: genes whose expression is important for fitness are selected to the leading strand because this reduces the duration of these interruptions. Our theory predicts that multi-gene operons, which are subject to longer interruptions, should be more strongly selected to the leading strand than singleton transcripts. We show that this is true even after controlling for the tendency for essential genes, which are strongly biased to the leading strand, to occur in operons. Our theory also predicts that other factors that are associated with strand bias should have stronger effects for genes that are in operons. We find that expression level and phylogenetic ubiquity are correlated with strand bias for both essential and non-essential genes, but only for genes in operons.
Abstract: Bacterial response to nitric oxide (NO) is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR, and NnrR; two-component systems NarXL and NarQP; NO-responsive activator NorR; and nitrite-sensitive repressor NsrR. Using comparative genomics approaches, we predict DNA-binding motifs for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA recognition motif. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria, including Clostridia, Thermotogales, and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides' metabolism, not only in most gamma- and beta-proteobacteria (including well-studied species such as Escherichia coli), but also in gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding motif. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon also includes two nitrite-responsive loci, nipAB (hcp-hcr) and nipC (dnrN), thus confirming the identity of the effector, i.e. nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include denitrification genes. As the result, we demonstrate considerable interconnection between various nitrogen-oxides-responsive regulatory systems for the denitrification and NO detoxification genes and evolutionary plasticity of this transcriptional network.
Abstract: At present, hundreds of microbial genomes have been sequenced, and hundreds more are currently in the pipeline. The Virtual Institute for Microbial Stress and Survival has developed a publicly available suite of Web-based comparative genomic tools (http://www.microbesonline.org) designed to facilitate multispecies comparison among prokaryotes. Highlights of the MicrobesOnline Web site include operon and regulon predictions, a multispecies genome browser, a multispecies Gene Ontology browser, a comparative KEGG metabolic pathway viewer, a Bioinformatics Workbench for in-depth sequence analysis, and Gene Carts that allow users to save genes of interest for further study while they browse. In addition, we provide an interface for genome annotation, which like all of the tools reported here, is freely available to the scientific community.
Abstract: Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress by using whole-genome DNA microarrays for S. oneidensis. Approximately 15% (n = 711) of the total predicted S. oneidensis genes (n = 4,648) represented on the microarray were significantly up- or downregulated (P < 0.05) over a 25-min period after shift to the heat shock temperature. As expected, the majority of the genes that showed homology to known chaperones and heat shock proteins in other organisms were highly induced. In addition, a number of predicted genes, including those encoding enzymes in glycolysis and the pentose cycle, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine kinases, and hypothetical proteins were induced. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed downregulated coexpression patterns in response to heat stress, as did genes encoding prophage and flagellar proteins. Finally, a putative regulatory site with high conservation to the Escherichia coli sigma32-binding consensus sequence was identified upstream of a number of heat-inducible genes.
Abstract: BACKGROUND: Relatively little is known about the genetic basis for the unique physiology of metal-reducing genera in the delta subgroup of the proteobacteria. The recent availability of complete finished or draft-quality genome sequences for seven representatives allowed us to investigate the genetic and regulatory factors in a number of key pathways involved in the biosynthesis of building blocks and cofactors, metal-ion homeostasis, stress response, and energy metabolism using a combination of regulatory sequence detection and analysis of genomic context. RESULTS: In the genomes of delta-proteobacteria, we identified candidate binding sites for four regulators of known specificity (BirA, CooA, HrcA, sigma-32), four types of metabolite-binding riboswitches (RFN-, THI-, B12-elements and S-box), and new binding sites for the FUR, ModE, NikR, PerR, and ZUR transcription factors, as well as for the previously uncharacterized factors HcpR and LysX. After reconstruction of the corresponding metabolic pathways and regulatory interactions, we identified possible functions for a large number of previously uncharacterized genes covering a wide range of cellular functions. CONCLUSIONS: Phylogenetically diverse delta-proteobacteria appear to have homologous regulatory components. This study for the first time demonstrates the adaptability of the comparative genomic approach to de novo reconstruction of a regulatory network in a poorly studied taxonomic group of bacteria. Recent efforts in large-scale functional genomic characterization of Desulfovibrio species will provide a unique opportunity to test and expand our predictions.
Abstract: Recent advances in high-throughput methods have provided us with a first glimpse of the overall structure of molecular interaction networks in biological systems. Ultimately, we expect that such information will change how we think about biological systems in a fundamental way. Instead of viewing the genetic parts list of an organism as a loose collection of biochemical activities, in the best case, we anticipate discrete networks of function to bridge the gap between genotype and phenotype, and to do so in a more profound way than the current qualitative classification of linked reactions into familiar pathways, such as glycolysis and the MAPK signal transduction cascades. At the present time, however, we are still far from a complete answer to the most basic question: what can we learn about biology by studying networks? Promising steps in this direction have come from such diverse approaches as mathematical analysis of global network structure, partitioning networks into functionally related modules and motifs, and even de novo design of networks. A complete picture will probably require integrating the data obtained from all of these approaches with modeling efforts at many different levels of detail.
Abstract: Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.
Abstract: Our understanding of the principles underlying the protein-folding problem can be tested by developing and characterizing simple models that make predictions which can be compared to experimental data. Here we extend our earlier model of folding free energy landscapes, in which each residue is considered to be either folded as in the native state or completely disordered, by investigating the role of additional factors representing hydrogen bonding and backbone torsion strain, and by using a hybrid between the master equation approach and the simple transition state theory to evaluate kinetics near the free energy barrier in greater detail. Model calculations of folding phi-values are compared to experimental data for 19 proteins, and for more than half of these, experimental data are reproduced with correlation coefficients between r=0.41 and 0.88; calculations of transition state free energy barriers correlate with rates measured for 37 single domain proteins (r=0.69). The model provides insight into the contribution of alternative-folding pathways, the validity of quasi-equilibrium treatments of the folding landscape, and the magnitude of the Arrhenius prefactor for protein folding. Finally, we discuss the limitations of simple native-state-based models, and as a more general test of such models, provide predictions of folding rates and mechanisms for a comprehensive set of over 400 small protein domains of known structure.
Abstract: The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.
Abstract: Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.
Abstract: BACKGROUND: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle. RESULTS: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition state, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta2 and beta4 remain associated. CONCLUSIONS: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta2-beta4 residual structure is surprising, because beta4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.
Abstract: Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the approximately 60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed beta-hairpins. We demonstrate that, as in the case of protein L, one of the two beta-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second beta-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain-main chain hydrogen bond in the second beta-turn slows the folding rate approximately 20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact beta-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated beta-hairpins.
Abstract: There has been considerable progress made over the past year in linking experimental and theoretical approaches to protein folding. Recent results from several independent lines of investigation suggest that protein folding mechanisms and landscapes are largely determined by the topology of the native state and are relatively insensitive to details of the interatomic interactions. This dependence on low-resolution structural features, rather than high-resolution detail, suggests that it should be possible to describe the fundamental physics of the folding process using relatively low-resolution models. Recent experiments have set benchmarks for testing new models and progress has been made in developing theoretical models for interpreting and predicting experimental results.
Abstract: Guided by recent experimental results suggesting that protein-folding rates and mechanisms are determined largely by native-state topology, we develop a simple model for protein folding free-energy landscapes based on native-state structures. The configurations considered by the model contain one or two contiguous stretches of residues ordered as in the native structure with all other residues completely disordered; the free energy of each configuration is the difference between the entropic cost of ordering the residues, which depends on the total number of residues ordered and the length of the loop between the two ordered segments, and the favorable attractive interactions, which are taken to be proportional to the total surface area buried by the ordered residues in the native structure. Folding kinetics are modeled by allowing only one residue to become ordered/disordered at a time, and a rigorous and exact method is used to identify free-energy maxima on the lowest free-energy paths connecting the fully disordered and fully ordered configurations. The distribution of structure in these free-energy maxima, which comprise the transition-state ensemble in the model, are reasonably consistent with experimental data on the folding transition state for five of seven proteins studied. Thus, the model appears to capture, at least in part, the basic physics underlying protein folding and the aspects of native-state topology that determine protein-folding mechanisms.
Abstract: We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded beta-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.
