Structural and Functional Roles of Coevolved Sites in Proteins

Understanding the residue covariations between multiple positions in protein families is very crucial and can be helpful for designing protein engineering experiments. These simultaneous changes or residue coevolution allow protein to maintain its overall structural-functional integrity while enabling it to acquire specific functional modifications. Despite the significant efforts in the field there is still controversy in terms of the preferable locations of coevolved residues on different regions of protein molecules, the strength of coevolutionary signal and role of coevolution in functional diversification.In this paper we study the scale and nature of residue coevolution in maintaining the overall functionality and structural integrity of proteins. We employed a large scale study to investigate the structural and functional aspects of coevolved residues. We found that the networks representing the coevolutionary residue connections within our dataset are in general of 'small-world' type as they have clustering coefficient values higher than random networks and also show smaller mean shortest path lengths similar and/or lower than random and regular networks. We also found that altogether 11% of functionally important sites are coevolved with any other sites. Active sites are found more frequently to coevolve with any other sites (15%) compared to protein (11%) and ligand (9%) binding sites. Metal binding and active sites are also found to be more frequently coevolved with other metal binding and active sites, respectively. Analysis of the coupling between coevolutionary processes and the spatial distribution of coevolved sites reveals that a high fraction of coevolved sites are located close to each other. Moreover, approximately 80% of charge compensatory substitutions within coevolved sites are found at very close spatial proximity (<or= 5A), pointing to the possible preservation of salt bridges in evolution.Our findings show that a noticeable fraction of functionally important sites undergo coevolution and also point towards compensatory substitutions as a probable coevolutionary mechanism within spatially proximal coevolved functional sites

J Comput Biol

Application of genetic distances to measure phenotypic relatedness is a challenging task, reflecting the complex relationship between genotype and phenotype. Accurate assessment of proximity among sequences with different phenotypic traits depends on how strongly the chosen distance is associated with structural and functional properties. In this study, we present a new distance measure Mutual Information and Entropy H (MIH) for categorical data such as nucleotide or amino acid sequences. MIH applies an information matrix (IM), which is calculated from the data and captures heterogeneity of individual positions as measured by Shannon entropy and coordinated substitutions among positions as measured by mutual information. In general, MIH assigns low weights to differences occurring at high entropy positions or at dependent positions. MIH distance was compared with other common distances on two experimental and two simulated data sets. MIH showed the best ability to distinguish cross-immunoreactive sequence pairs from non-cross-immunoreactive pairs of variants of the hepatitis C virus hypervariable region 1 (26,883 pairwise comparisons), and Major Histocompatibility Complex (MHC) binding peptides (|\u2009=\u2009181) from non-binding peptides (|\u2009=\u2009129). Analysis of 74 simulated RNA secondary structures also showed that the ratio between MIH distance of sequences from the same RNA structure and MIH of sequences from different structures is three orders of magnitude greater than for Hamming distances. These findings indicate that lower MIH between two sequences is associated with greater probability of the sequences to belong to the same phenotype. Examination of rule-based phenotypes generated in silico showed that (1) MIH is strongly associated with phenotypic differences, (2) IM of sequences under selection is very different from IM generated under random scenarios, and (3) IM is robust to sampling. In conclusion, MIH strongly approximates structural/functional distances and should have important applications to a wide range of biological problems, including evolution, artificial selection of biological functions and structures, and measuring phenotypic similarity.CC999999/ImCDC/Intramural CDC HHSUnited States

Understanding The Intra And Inter-Cellular Interaction Complexities And Flexibilities Using Systems And Sequence Analysis Approach

The present thesis work has been undertaken to gain an understanding of intra-cellular or inter-cellular interactions between bio-molecular entities utilizing either a systems analysis based perspective or different sequence analysis approaches. During this study different principles likely to be prevalent among intra-cellular and inter-cellular interactions have been studied with the help of computational approaches. Broadly, the complexities in intra-cellular interactions have been studied by determining the effect of perturbations such as over-expression or down-regulation of a key regulator on the intra-cellular interaction network architecture or its components. In particular, network analysis of regulatory network proteins in association with the intra-cellular proteinprotein interaction network, led to a key observation that topologically important effector proteins in the regulatory network could be important signaling proteins. Identification of such important effector proteins essential for the regulatory network integrity of a key regulator may be performed by network analysis. It is likely that alterations in these important effector proteins may lead to disruptions in cellular physiology and as such in this manner probable disease associated entities can be determined. Alternately, the flexibility among protein-protein interactions has been studied by analyzing homologous sequence families of interacting proteins with the help of information theory based measures like mutual information and Bhattacharyya co-efficient. Since interacting proteins may co-evolve, co-variation may allow the preservation of a functional interaction between co-evolving proteins and interdependent residue pair alterations may occur as a result of evolutionary pressure. Analysis of molecular co-evolution in inter-cellular protein interaction complexes determined that co-evolutionary pairings may be present among interface and noninterface residue pairs and such positions are likely to be crucial for a functional interaction between these sets of proteins. Therefore, utilising information contained in biological sequences, co-evolutionary pairings involving structurally or functionally crucial residue positions in disease associated inter-cellular protein-protein interaction complexes were predicted. Thus, different computational approaches have been utilised to study a particular hypothesis in a disease scenario in order to delineate certain themes prevalent in intra-cellular or inter-cellular interactions among bio-molecular entities while predicting disease associated entities or studying interaction patterns among them

Genome Mining, Isolation and Characterization of Novel Lasso Peptides and Their Utilization in Drug Development

Lasso peptides are a class of natural products that belong to the family of ribosomally‑assembled and posttranslationally‑modified peptides. They are defined by an unique structural motif referred to as the so‑called lariat knot, whose name is derived from the fact that this topology is reminiscent of the knot found in the noose of a lasso. This structure is achieved by the presence of an N‑terminal macrolactam ring that is threaded by the C‑terminal tail of the peptide. The fold in these molecules is then conserved by non‑covalent interactions in the form of bulky amino acids located above and below the macrolactam ring, in this way entrapping the tail inside of the ring. What makes these compounds of interest for research is that their structure, even though it is maintained merely by sterical interactions, often exhibits a tremendous stability against thermal, chemical and proteolytic degradation. Still, up to now little is known about the general function of these compounds for their producing organisms, although there are some interesting biological activities attributed to some of the previously reported lasso peptides. To obtain more information about their physico‑chemical properties, their biosynthesis and to get an idea what role they might play in nature, the primary subject of this thesis was the directed genome mining for and the subsequent isolation and characterization of novel lasso peptides. The results of these projects were published in several studies that will be shown and discussed in the course of this thesis. Amongst other findings, these studies not only include the discovery of a multitude of novel lasso peptides, but through the thorough analysis and characterization of these compounds, several former assumptions of this research area could be overhauled and updated. In addition to this, the bioinformatic data gathered during our genome mining studies furthermore uncovered interesting facts about the distribution of lasso peptides amongst bacteria and about the existence of different subgroups of biosynthetic gene cluster arrangements, which could facilitate future research directed towards identifiying the concrete functions of these compounds. Furthermore, it was also investigated if these compounds are suitable scaffolds for drug development via epitope grafting approaches. In this regard, a previously reported bioactive lasso graft was used as the basis to show that such compounds can indeed be further optimized and improved upon by rational approaches that utilize the information obtained from research done with simple linear or cyclic peptides that are, in contrast to lasso peptides, easily accessible by synthetic means

Molecular modelling and biological implications of non-canonical structures in RNA viruses and long non-coding RNAs

Guanine quadruplexes folding in single-strand DNA have been extensively studied for some time. But far less attention has been paid to systems arising in duplex DNA or RNA. Furthermore, the literature shows an apparent absence of quadruplex systems generated exclusively by other nucleic acid bases. With guanine quadruplexes (G4s) as a benchmark, a comparison is made here with equivalent complexes folding in DNA and RNA when derived from cytosine, thymine and adenine. Molecular dynamics simulations determined cytosine and thymine models in DNA as relatively fragile systems, but all non-guanine RNA models were found to be stable into biologically relevant times. Uniquely biplanar or triplanar adenine quadruplexes (A4s) have ostensibly not been described. Adenine models constructed for this work were resolved in silico with stabilities comparable to known guanine equivalents. These complexes achieved further significance with the advent of SARS-CoV-2. RNA viruses are characterised by a poly-adenylated structure capping the genomic terminus. This poly(A) tail is crucial to a cascade of viral replicative activity occurring both extra- and intra-cellular during infection. As a route to proposing potential chemotherapy, this study suggests simple biplanar A4s may fold in this poly-adenylated domain. Notably, mRNA configured as a biplanar A4, shows less dynamic activity than DNA equivalents. This observation may be especially relevant in a physiological context. In contrast to well-characterised guanine quadruplexes, co-ordination with cations appears not to impact on stability. These conclusions may apply to SARS-CoV-2, its variants and other pathogenic RNA viruses. The thesis also infers models of potential interactions between small nucleolar RNAs and long non-coding RNAs may have biological relevance. Non-canonical base-pairing in some instances suggests a molecular mechanism for dysfunction in the development of the embryonic pre-frontal cortex

Algorytmy i modele do analizy struktur białkowych

In this work we present several algorithmic approaches designed to help researchers in the study of various orders of protein structure. To facilitate the study of molecular sequence evolution we present an algorithm for multiple alignment of sequence profiles, describe a tool that can be used to study the relationship between residue co-evolution and structure, and a database of structures modeled based co-evolutionary approach. On the structure side, a new algorithm for knot type assignment in biological molecules is introduced, a database of linked protein structures is described, and a method of fixing structure models in a topologically-conscious way is presented. Additionally, folding pathways of several newly discovered knotted proteins are proposed, and the influence of coevolution-based interactions of folding simulations discussed.Niniejsza rozprawa doktorska omawia szereg metod mających zastosowanie w badaniu białek na wielu płaszczyznach. Pierwszy rozdział wprowadza nowy algorytm pozwalający na określenie typu węzła w biocząsteczkach. Drugi rozdział poświęcony jest ewolucji sekwencji molekularnych. Na początku opisany jest nowy algorytm do multiuliniawiania profili sekwencyjnych oraz jego zastosowanie w badaniu ewolucji białek membranowych zawierających zduplikowane domeny. Następnie przedstawione jest narzędzie pozwalające na badanie związków między koewolucją sekwencji (znalezioną poprzez metodę Direct Coupling Analysis), a strukturą cząsteczki, oraz baza danych struktur wymodelowanych na podstawie koewolucji sekwencji. Wreszcie przedstawione jest zastosowanie oddziaływań wskazanych przez koewolucję w symulacjach zwijania białek. Ostatni rozdział poświęcony jest badaniom nietrywialnych topologicznie struktur białek, poprzez bazę danych struktur zawierających linki oraz metodę naprawy modeli struktur z zachowaniem właściwej topologii. Na koniec przedstawione są propozycje ścieżek zwijania dla nowopoznanych struktur białek z węzłami

The Mechanisms of DNA Replication

DNA replication is a fundamental part of the life cycle of all organisms. Not surprisingly many aspects of this process display profound conservation across organisms in all domains of life. The chapters in this volume outline and review the current state of knowledge on several key aspects of the DNA replication process. This is a critical process in both normal growth and development and in relation to a broad variety of pathological conditions including cancer. The reader will be provided with new insights into the initiation, regulation, and progression of DNA replication as well as a collection of thought provoking questions and summaries to direct future investigations

NMR study of CyaY and YFHJ, two proteins involved in iron metabolism

From the Summary: In this thesis we apply Nuclear Magnetic Resonance to gain insights into the structures and functions of the two proteins CyaY and YFHJ. The overall material is organised in eight chapters, an introduction and a conclusion. Chapter 1: In this chapter the basis of Nuclear Magnetic Resonance are provided, with reference to its application to the study of biological macromolecules Chapter 2: This chapter describes the 2D and 3D NMR experiments that allow the assignment of a protein\u2019s spectra. Chapter 3: Here a short description of the bioinformatic tools and software packages available to extract information on macromolecules is provided. The material is organised following the proteins\u2019 level of structure it refers to: first there is a short description of the web databases and the programs that can be used to extract information from the primary sequence of a protein, then an elucidation of the programs useful to analyse the secondary structure and some details on the one applied in this study to calculate the 3D structure; finally, the available tools for structure comparison and refinement are reported. Chapter 4: This chapter summarises what is known about Friedreich\u2019s ataxia and frataxin. Chapter 5: Here the study of CyaY iron binding site by means of NMR is presented. In the first part of the chapter the problems and advantages of using paramagnetic probes in NMR are briefly discussed. Chapter 6: This chapter reviews the systems involved in iron-sulphur clusters\u2019 biosynthesis with particular attention to the proteins encoded by the bacterial Iron Sulphur Cluster operon. Chapter 7: An elucidation of the 3D structure determination process of the protein YFHJ is reported. In the first part circular dichroism and its application in biomolecular studies are discussed. Chapter 8: This chapter illustrates the functional characterisation of the protein YFHJ by means of NMR, mass spectrometry and fluorescence polarisation. The techniques used are elucidated in the initial paragraphs

Biosynthesis and regulation of cyclic lipopeptides in Pseudomonas fluorescens

Cyclic lipopeptides (CLPs) are surfactant and antibiotic metabolites produced by a variety of bacterial genera. For the genus Pseudomonas, many structurally different CLPs have been identified. CLPs play an important role in surface motility of Pseudomonas strains, but also in virulence and attachment/detachment to and from surfaces. In this Ph.D. thesis project, two new CLP biosynthesis clusters were identified in Pseudomonas fluorescens and fully sequenced. In P. fluorescens strain SBW25, the viscosin biosynthesis cluster was identified by bioinformatic analyses of the genome followed by genetic and chemical analyses. For P. fluorescens strain SS101, the genes for massetolide biosynthesis were identified via random mutagenesis followed by cloning, sequencing and chemical analyses. Biosynthesis of viscosin and massetolide is governed by three nonribosomal peptide synthetase (NRPS) genes, designated viscABC and massABC, respectively. The viscosin and massetolide biosynthesis gene clusters are very similar, but different from CLP gene clusters described for other Pseudomonas as the viscA and massA genes are physically disconnected from viscBC and massBC, respectively. Viscosin differs from massetolide A only at position number four in the peptide moiety, which is a valine in viscosin and an isoleucine in massetolide A. Because of the modular structure of the NRPSs and the co-linearity of the assembly process, transfer of the mass genes of strain SS101 into strain SBW25 resulted in the production of both massetolide A and viscosin, demonstrating that the assembly line for CLP biosynthesis in Pseudomonas can be altered leading to the production of non-native products. Compared to the understanding of CLP biosynthesis, not so much is known about the regulation. This thesis shows that the GacA/GacS two-component system regulates massetolide and viscosin biosynthesis in strains SS101 and SBW25, respectively. No indications were found that massetolide or viscosin biosynthesis is regulated by quorum sensing via N-acylhomoserine lactones. Site-directed mutagenesis of the LuxR-type regulator genes luxR-vA and luxR-vBC flanking the viscosin biosynthesis cluster resulted in a loss of viscosin production, indicating that both LuxR-type transcriptional regulators are important for viscosin biosynthesis in strain SBW25. Phylogenetic analyses further suggested that these LuxR-type transcriptional regulators do not contain the autoinducerbinding domain found for the quorum sensing-associated LuxR regulator in Vibrio fischeri. Instead, the LuxR-type regulator genes flanking the massetolide and viscosin biosynthesis genes are closely related to the LuxR-type regulators identified for syringomycin/ syringopeptin biosynthesis and appear to belong to a separate LuxR-type regulator subfamily, different from the autonomous effector domain protein GerE. Via random mutagenesis and subsequent screening for massetolide-deficient mutants, also other regulator genes were identified including clpP. ClpP is a serine protease that plays a crucial role in intracellular refolding and degradation of proteins, which is an essential process for the viability of cells. ClpP was shown to affect transcription of luxR-mA, thereby regulating transcription of the massetolide biosynthesis genes. Results further suggested that, at the transcriptional level, ClpPmediated regulation of massetolide biosynthesis operates independently from regulation by the GacA/GacS two-component system. In conclusion, the results of this thesis led to the identification of several genes and previously unknown pathways involved in regulation of CLP biosynthesis and highlighted the complexity of the signaling cascades underlying CLP biosynthesis in Pseudomonas. CLPs have diverse functions for the producing bacterial strains, including a role in motility, biofilm formation, antimicrobial activity and virulence. Also in establishment and persistence in plant environments, CLPs were shown to confer a competitive advantage. A new function of CLPs, identified in a collaboration with Mark Mazzola (USDA) and presented in this thesis, is their protective effects against predation by protozoa. In vitro assays showed that both massetolide and viscosin can lyse the trophozoites of Naeglaria americana and that wild type strains SS101 and SBW25 were substantially less sensitive to protozoan grazing than their CLP-deficient mutants. Also in soil containing N. americana, population densities of wild type strains SS101 and SBW25 were significantly higher compared to the massetolide and viscosin-deficient mutants, showing that CLP production confers a competitive advantage in survival in complex environments. Moreover, transcription of the CLP-biosynthesis genes increased significantly upon protozoan grazing, indicating that the Pseudomonas strains sense the protozoa and react by producing CLPs as defense compounds. Which signal triggers the induction of the CLP biosynthesis genes is not known yet and currently under investigation. Based on these results, we postulate that CLPs are an important component of the preingestional defense mechanisms of bacteria against protozoan predation, not only due to their lytic effects on protozoa, but also because CLPs contribute to evasion of protozoan grazing via altered cell surface properties, swimming and swarming, and microcolony and biofilm formation. <br/

Study on Two Chitin-Binding Proteins From Vibrio Parahaemolyticus.

Two chitin-binding proteins from Vibrio parahaemolyticus are secreted into the culture medium after the addition of chitin or oligomers of N-acetylglucosamine, but only to lower levels of expression with a few other saccharides. The first protein exhibited a very salt-resistant chitinase activity with activity maxima at pH 6 and 9. The enzyme is stable for 30 minutes at 40\sp\circC, and maximal activity is reached at 50\sp\circC. The chitinase hydrolyzes optimally chitin oligomer substrates with degree of polymerization of 4 and higher. The hydrolytic activity upon the chitotrimer is low; the dimer cannot be hydrolyzed by the enzyme. When incubated with chitotrimer, the protein exhibits transglycosylase activity. The chitinase from V. parahaemolyticus shows a strong sequence homology with chitinases from Serratia marcescens and Bacillus circulans. The other protein purified from Vibrio parahaemolyticus culture supernatant fractions is a chitin-binding protein with an apparent molecular weight of 134,000. The protein was either purified by conventional ammonium sulfate precipitation followed by fractionation on a gel filtration column, or by affinity chromatography on regenerated chitin, followed by differential elution with increasing concentrations of guanidine hydrochloride. Samples of this protein eluted with guanidine hydrochloride retained their binding capacity after dialysis. The protein could also be specifically eluted from chitin with a concentrated solution of chitin oligomers. The N-terminal amino acid sequence of the protein showed no strong homology to any known protein sequences in the GeneBank data bank, suggesting that it may be a novel carbohydrate-binding protein tentatively named chitovibrin. Chitovibrin exhibits similar chromatographic characteristics to chitinase, but polyclonal antibodies raised against chitinase do not cross-react with chitovibrin. The function of chitovibrin has yet to be determined