Role of disulfide bonds in stabilizing the conformation of selected enzymes : an approach based on divergence entropy applied to the structure of hydrophobic core in proteins

One of the factors responsible for tertiary structural stabilization in proteins is the presence of the hydrophobic core—a result of hydrophobic interactions within the protein body. In some proteins (especially extracellular ones) additional stabilization is provided by covalent bonds between selected Cys residues, commonly referred to as disulfide bonds. The mutual interplay of both factors and their respective contributions to stabilization are the focus of this work. The assessment of the effects of disulfide bonds isinterpreted by Fuzzy Oil Drop (FOD) model in which individual polypeptide chain fragments (including fragments which participate in SS bonds) can be evaluated in the context of their influence upon tertiary structural stabilization by comparing their corresponding theoretical and idealized hydrophobicity density distributions. The proteins were identified with both factors reinforcing each other, as well as proteins where they seem to counteract each other. The analysis presents a number of enzymes, including ribonuclease, lysozyme, disulfide isomerase and phospholipase

Sequence-to-structure relation in proteins-amyloidogenic proteins with chameleon sequences

The existence of polypeptide chain fragments in which identical sequences translate into different secondary folds gives rise to questions concerning the structural variability associated with amyloidogenesis. In this paper the structural contribution of identical sequences to a common hydrophobic core is assessed on the basis of the fuzzy oil drop model. The model compares the observed hydrophobicity density distribution in a protein molecule to its idealized counterpart, where all hydrophilic residues are exposed on the surface while all hydrophobic residues are internalized. The conformational variability of such fragments is thought to be associated with their role: they either participate in the formation of a stable core, or become involved in mediating the protein’s biological function. The fuzzy oil drop model provides clues as to the role of chameleon sequences in prions, seen as potential loci of conformational changes resulting in amyloidogenesis

Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families

International audienceBackground: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue.Results: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues.Conclusion: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function

Protein stability in a proteomic perspective

Dissertation presented to obtain the Ph.D degree in BiochemistryThis work involved the identification and analysis of the properties of the most stable proteins present within proteomes, aiming at obtaining a general perspective of the factors that determine protein stability. As models we have focused on ensembles of proteins with high intrinsic stability, and for this purpose we have studied proteomes from the hyperthermophilic archaeon Sulfolobus solfataricus and Sulfurisphaera sp., whose properties were compared to those of the mesophilic bacterium Escherichia coli.(...

Evolution-guided Engineering of Alpha/Beta Hydrolases

University of Minnesota Ph.D. dissertation. June 2017. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Romas Kazlauskas. 1 computer file (PDF); xx, 321 pages.This work applies principles from evolution to engineering enzyme properties. Specifically, by examining the phylogeny and evolved sequence diversity in a group of α/β-hydrolase fold enzymes from plants, we are able to engineer proteins with broader chemoselectivity, altered enantioselectivity, and increased stability. A number of ancestral α/β-hydrolases fold proteins were reconstructed in one set of experiments. These were more likely than related modern proteins to have relaxed chemoselectivities and, in one case, was more useful for synthesizing medicinally important molecules. Relative to modern enzymes, ancestral enzymes near functional branch points could catalyze more esterase and hydroxynitrile lyase reactions, as well as a number of other types of reactions: decarboxylation, Michael addition, γ-lactam hydrolysis, and 1,5-diketone hydrolysis. This finding helps to demonstrate the important role that enzyme promiscuity plays in the evolution of new enzymes. Additional experiments and structural analysis on one of these reconstructed ancestral enzymes, the early hydroxynitrile lyase HNL1 found that it is both more thermostable and more promiscuous than its modern relatives, HbHNL and MeHNL. X-ray crystallographic studies revealed, counterintuitively, that larger amino acids in the active site of the ancestor actually increased the size of the substrate binding pocket relative to modern relatives. To take advantage of the promiscuity observed in HNL1, it was used in the asymmetric synthesis of a precursor for the important pharmaceutical propranolol. Another set of experiments altered enantioselectivity by making phylogenetically informed mutations. The active sites from two related hydroxynitrile lyases, HbHNL and AtHNL, were modified to resemble their last common ancestor. This resulted in altered enantioselectivity, and in the case of AtHNL, reversed enantioselectivity. Surprisingly modeling suggested that some of these mutants use a previously undescribed mechanism. This may have been the extinct ancestral mechanism that served as an evolutionary stepping stone that allowed descendant lineages to diverge to either the S-HNL mechanism used by HbHNL, or the R-HNL mechanism used by AtHNL. A final set of experiments used a variety of methods to identify stabilizing mutations in another plant α/β-hydrolase, SABP2. All of the methods were able to identify stabilizing mutations. The most stabilizing mutations were identified by methods that used no structural information. Random mutagenesis identified highly stabilizing mutations, but required screening thousands of mutants. The most efficient approaches were found to be those that used sequence information from either one stable homolog, or the consensus of many homologs, to identify potential stabilizing mutations. Residues that evolution has conserved are often important for stabilizing a protein. We created a software application, Consensus Finder, to automate the process of identifying stabilizing mutations by consensus

The Complex Role of Sequence and Structure in the Stability and Function of the TIM Barrel Proteins

Sequence divergence of orthologous proteins enables adaptation to a plethora of environmental stresses and promotes evolution of novel functions. As one of the most common motifs in biology capable of diverse enzymatic functions, the TIM barrel represents an ideal model system for mapping the phenotypic manifestations of protein sequence. Limits on evolution imposed by constraints on sequence and structure were investigated using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Exploration of fitness landscapes of phylogenetically distant orthologs provides a strategy for elucidating the complex interrelationship in the context of a protein fold. Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape. These results suggest that fitness landscapes of point mutants can be successfully translocated in sequence space, where knowledge of one landscape may be predictive for the landscape of another ortholog. Analysis of a surprising class of beneficial mutations in all three IGPS orthologs pointed to a long-range allosteric pathway towards the active site of the protein. Biophysical and biochemical analyses provided insights into the molecular mechanism of these beneficial fitness effects. Epistatic interactions suggest that the helical shell may be involved in the observed allostery. Taken together, knowledge of the fundamental properties of the TIM protein architecture will provide new strategies for de novo protein design of a highly targeted protein fold

Filamentous aggregates of tau proteins fulfil standard amyloid criteria provided by the fuzzy oil drop (FOD) model

Abnormal filamentous aggregates that are formed by tangled tau protein turn out to be classic amyloid fibrils, meeting all the criteria defined under the fuzzy oil drop model in the context of amyloid characterization. The model recognizes amyloids as linear structures where local hydrophobicity minima and maxima propagate in an alternating manner along the fibril’s long axis. This distribution of hydrophobicity differs greatly from the classic monocentric hydrophobic core observed in globular proteins. Rather than becoming a globule, the amyloid instead forms a ribbonlike (or cylindrical) structure

Application of divergence entropy to characterize the structure of the hydrophobic core in DNA interacting proteins

The fuzzy oil drop model, a tool which can be used to study the structure of the hydrophobic core in proteins, has been applied in the analysis of proteins belonging to the jumonji group—JARID2, JARID1A, JARID1B and JARID1D—proteins that share the property of being able to interact with DNA. Their ARID and PHD domains, when analyzed in the context of the fuzzy oil drop model, are found to exhibit structural variability regarding the status of their secondary folds, including the $\beta$ -hairpin which determines their biological function. Additionally, the structure of disordered fragments which are present in jumonji proteins (as confirmed by the DisProt database) is explained on the grounds of the hydrophobic core model, suggesting that such fragments contribute to tertiary structural stabilization. This conclusion is supported by divergence entropy measurements, expressing the degree of ordering in each protein’s hydrophobic core