Protein folding on the ribosome studied using NMR spectroscopy

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome-nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity

A Minimal TrpRS Catalytic Domain Supports Sense/Antisense Ancestry of Class I and II Aminoacyl-tRNA Synthetases

The emergence of polypeptide catalysts for amino acid activation, the slowest step in protein synthesis, poses a significant puzzle associated with the origin of biology. This problem is compounded as the 20 contemporary aminoacyl-tRNA synthetases belong to two quite distinct families. We describe here the use of protein design to show experimentally that a minimal class I aminoacyl-tRNA synthetase active site might have functioned in the distant past. We deleted the anticodon binding domain from tryptophanyl-tRNA synthetase and fused the discontinuous segments comprising its active site. The resulting 130 residue minimal catalytic domain activates tryptophan. This residual catalytic activity constitutes the first experimental evidence that the conserved class I signature sequences, HIGH and KMSKS, might have arisen in-frame, opposite motifs 2 and 1 from class II, as complementary sense and antisense strands of the same ancestral gene

Detection of single-nucleotide polymorphism Gap junction protein Beta-2 genes in deaf schoolchildren of javanese population in Surabaya, Indonesia

Background: Genetic factors account for about 50%–75% responsible for hearing loss. The existence of single-nucleotide polymorphism (SNP) as genetic factors that affect hereditary hearing loss. The widely studied SNP was the gap junction protein beta-2 (GJB2) gene encoding the gap junction beta-2 protein (connexin26) that found in cochlea and required to convert sound waves into electrical nerve impulse. This study was aimed to detect the SNP GJB2 gene of hereditary hearing loss patients from Javanese population in Surabaya, Indonesia. Methods: The design of this study was a cross-sectional, analytic observational. The participant was taken randomly among the students from a deaf School in Surabaya. The questionnaire was completed by the parents of the deaf children. Blood sampling was taken from venous peripheral blood. DNA was extracted and amplified on GJB2 gene area by polymerase chain reaction (PCR). The positive results of PCR were processed further for sequencing. The sequencing results were analyzed to detect the GJB2 gene SNP with reference sequence/rs-80338939. Results: A total of 22 children participated in this study; all were profound sensorineural hearing loss (SNHL). The hereditary hearing loss was obtained with fewer in five children (22.73%), who had a history of hearing loss in their family. It was compared to 17 children (77.27%) who had no family history of hearing loss. It was found that the nucleotide variation in nucleotide number 8473 of GJB2 gene as much as 3 (13.64%) out of 22 children in hereditary hearing loss patients in Deaf School Type B Surabaya. Conclusions: This study did not found any SNP GJB2 gene (rs-80338939) of hereditary hearing loss patients from the Javanese population in Surabaya, Indonesia. There was the nucleotide substitution G to A in nucleotide number 8473 of GJB2 gene, which indicated the change of amino acid code genetic code table (valine) to amino acid code genetic code table (isoleucine). It may as the cause of SNHL

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

MOLECULAR DYNAMICS ANALYSIS OF THE BEHAVIOUR OF PROLYL OLIGOPEPTIDASE (POP) IN THE PRESENCE OF Z-PRO-PROLINAL INHIBITOR

Prolyl Oligopeptidase (POP), a member of the prolyl endopeptidase family, has a role in several neurological disorders. Its primary function is to cleave an oligopeptidase, including neuroactive peptides. On the other hand POP with a Z-Pro-prolinal (ZPP) inhibitor may revert memory loss from neurological disorders, amnesic agents and aging. Here, the crystal structure of POP protein with ZPP inhibitor (Protein Data Bank PDB) and without ZPP inhibitor is studied using classical molecular dynamics simulations and the POP-ZPP complex behaviour is compared with pure POP. The basic analysis of the structures, included measuring radius of gyration and root mean square deviation which proved that POP structure with non-bonded ZPP and without ZPP are stable and maintain their structure over the entire simulation time. Moreover, principal component analysis (PCA) is used to analyze the motions of the structures by extracting the normal modes of motions in POP with and without presence of ZPP inhibitor

Protein-protein interactions: impact of solvent and effects of fluorination

Proteins have an indispensable role in the cell. They carry out a wide variety of structural, catalytic and signaling functions in all known biological systems. To perform their biological functions, proteins establish interactions with other bioorganic molecules including other proteins. Therefore, protein-protein interactions is one of the central topics in molecular biology. My thesis is devoted to three different topics in the field of protein-protein interactions. The first one focuses on solvent contribution to protein interfaces as it is an important component of protein complexes. The second topic discloses the structural and functional potential of fluorine's unique properties, which are attractive for protein design and engineering not feasible within the scope of canonical amino acids. The last part of this thesis is a study of the impact of charged amino acid residues within the hydrophobic interface of a coiled-coil system, which is one of the well-established model systems for protein-protein interactions studies. I. The majority of proteins interact in vivo in solution, thus studies of solvent impact on protein-protein interactions could be crucial for understanding many processes in the cell. However, though solvent is known to be very important for protein-protein interactions in terms of structure, dynamics and energetics, its effects are often disregarded in computational studies because a detailed solvent description requires complex and computationally demanding approaches. As a consequence, many protein residues, which establish water-mediated interactions, are neither considered in an interface definition. In the previous work carried out in our group the protein interfaces database (SCOWLP) has been developed. This database takes into account interfacial solvent and based on this classifies all interfacial protein residues of the PDB into three classes based on their interacting properties: dry (direct interaction), dual (direct and water-mediated interactions), and wet spots (residues interacting only through one water molecule). To define an interaction SCOWLP considers a donor–acceptor distance for hydrogen bonds of 3.2 Å, for salt bridges of 4 Å, and for van der Waals contacts the sum of the van der Waals radii of the interacting atoms. In previous studies of the group, statistical analysis of a non-redundant protein structure dataset showed that 40.1% of the interfacial residues participate in water-mediated interactions, and that 14.5% of the total residues in interfaces are wet spots. Moreover, wet spots have been shown to display similar characteristics to residues contacting water molecules in cores or cavities of proteins. The goals of this part of the thesis were: 1. to characterize the impact of solvent in protein-protein interactions 2. to elucidate possible effects of solvent inclusion into the correlated mutations approach for protein contacts prediction To study solvent impact on protein interfaces a molecular dynamics (MD) approach has been used. This part of the work is elaborated in section 2.1 of this thesis. We have characterized properties of water-mediated protein interactions at residue and solvent level. For this purpose, an MD analysis of 17 representative complexes from SH3 and immunoglobulin protein families has been performed. We have shown that the interfacial residues interacting through a single water molecule (wet spots) are energetically and dynamically very similar to other interfacial residues. At the same time, water molecules mediating protein interactions have been found to be significantly less mobile than surface solvent in terms of residence time. Calculated free energies indicate that these water molecules should significantly affect formation and stability of a protein-protein complex. The results obtained in this part of the work also suggest that water molecules in protein interfaces contribute to the conservation of protein interactions by allowing more sequence variability in the interacting partners, which has important implications for the use of the correlated mutations concept in protein interactions studies. This concept is based on the assumption that interacting protein residues co-evolve, so that a mutation in one of the interacting counterparts is compensated by a mutation in the other. The study presented in section 2.2 has been carried out to prove that an explicit introduction of solvent into the correlated mutations concept indeed yields qualitative improvement of existing approaches. For this, we have used the data on interfacial solvent obtained from the SCOWLP database (the whole PDB) to construct a “wet” similarity matrix. This matrix has been used for prediction of protein contacts together with a well-established “dry” matrix. We have analyzed two datasets containing 50 domains and 10 domain pairs, and have compared the results obtained by using several combinations of both “dry” and “wet” matrices. We have found that for predictions for both intra- and interdomain contacts the introduction of a combination of a “dry” and a “wet” similarity matrix improves the predictions in comparison to the “dry” one alone. Our analysis opens up the idea that the consideration of water may have an impact on the improvement of the contact predictions obtained by correlated mutations approaches. There are two principally novel aspects in this study in the context of the used correlated mutations methodology : i) the first introduction of solvent explicitly into the correlated mutations approach; ii) the use of the definition of protein-protein interfaces, which is essentially different from many other works in the field because of taking into account physico-chemical properties of amino acids and not being exclusively based on distance cut-offs. II. The second part of the thesis is focused on properties of fluorinated amino acids in protein environments. In general, non-canonical amino acids with newly designed side-chain functionalities are powerful tools that can be used to improve structural, catalytic, kinetic and thermodynamic properties of peptides and proteins, which otherwise are not feasible within the use of canonical amino acids. In this context fluorinated amino acids have increasingly gained in importance in protein chemistry because of fluorine's unique properties: high electronegativity and a small atomic size. Despite the wide use of fluorine in drug design, properties of fluorine in protein environments have not been yet extensively studied. The aims of this part of the dissertation were: 1. to analyze the basic properties of fluorinated amino acids such as electrostatic and geometric characteristics, hydrogen bonding abilities, hydration properties and conformational preferences (section 3.1) 2. to describe the behavior of fluorinated amino acids in systems emulating protein environments (section 3.2, section 3.3) First, to characterize fluorinated amino acids side chains we have used fluorinated ethane derivatives as their simplified models and applied a quantum mechanics approach. Properties such as charge distribution, dipole moments, volumes and size of the fluoromethylated groups within the model have been characterized. Hydrogen bonding properties of these groups have been compared with the groups typically presented in natural protein environments. We have shown that hydrogen and fluorine atoms within these fluoromethylated groups are weak hydrogen bond donors and acceptors. Nevertheless they should not be disregarded for applications in protein engineering. Then, we have implemented four fluorinated L-amino acids for the AMBER force field and characterized their conformational and hydration properties at the MD level. We have found that hydrophobicity of fluorinated side chains grows with the number of fluorine atoms and could be explained in terms of high electronegativity of fluorine atoms and spacial demand of fluorinated side-chains. These data on hydration agrees with the results obtained in the experimental work performed by our collaborators. We have rationally engineered systems that allow us to study fluorine properties and extract results that could be extrapolated to proteins. For this, we have emulated protein environments by introducing fluorinated amino acids into a parallel coiled-coil and enzyme-ligand chymotrypsin systems. The results on fluorination effect on coiled-coil dimerization and substrate affinities in the chymotrypsin active site obtained by MD, molecular docking and free energy calculations are in strong agreement with experimental data obtained by our collaborators. In particular, we have shown that fluorine content and position of fluorination can considerably change the polarity and steric properties of an amino acid side chain and, thus, can influence the properties that a fluorinated amino acid reveals within a native protein environment. III. Coiled-coils typically consist of two to five right-handed α-helices that wrap around each other to form a left-handed superhelix. The interface of two α-helices is usually represented by hydrophobic residues. However, the analysis of protein databases revealed that in natural occurring proteins up to 20% of these positions are populated by polar and charged residues. The impact of these residues on stability of coiled-coil system is not clear. MD simulations together with free energy calculations have been utilized to estimate favourable interaction partners for uncommon amino acids within the hydrophobic core of coiled-coils (Chapter 4). Based on these data, the best hits among binding partners for one strand of a coiled-coil bearing a charged amino acid in a central hydrophobic core position have been selected. Computational data have been in agreement with the results obtained by our collaborators, who applied phage display technology and CD spectroscopy. This combination of theoretical and experimental approaches allowed to get a deeper insight into the stability of the coiled-coil system. To conclude, this thesis widens existing concepts of protein structural biology in three areas of its current importance. We expand on the role of solvent in protein interfaces, which contributes to the knowledge of physico-chemical properties underlying protein-protein interactions. We develop a deeper insight into the understanding of the fluorine's impact upon its introduction into protein environments, which may assist in exploiting the full potential of fluorine's unique properties for applications in the field of protein engineering and drug design. Finally we investigate the mechanisms underlying coiled-coil system folding. The results presented in the thesis are of definite importance for possible applications (e.g. introduction of solvent explicitly into the scoring function) into protein folding, docking and rational design methods. The dissertation consists of four chapters: ● Chapter 1 contains an introduction to the topic of protein-protein interactions including basic concepts and an overview of the present state of research in the field. ● Chapter 2 focuses on the studies of the role of solvent in protein interfaces. ● Chapter 3 is devoted to the work on fluorinated amino acids in protein environments. ● Chapter 4 describes the study of coiled-coils folding properties. The experimental parts presented in Chapters 3 and 4 of this thesis have been performed by our collaborators at FU Berlin. Sections 2.1, 2.2, 3.1, 3.2 and Chapter 4 have been submitted/published in peer-reviewed international journals. Their organization follows a standard research article structure: Abstract, Introduction, Methodology, Results and discussion, and Conclusions. Section 3.3, though not published yet, is also organized in the same way. The literature references are summed up together at the end of the thesis to avoid redundancy within different chapters

Codon Bias Patterns of E.coliE.coli's Interacting Proteins

Synonymous codons, i.e., DNA nucleotide triplets coding for the same amino acid, are used differently across the variety of living organisms. The biological meaning of this phenomenon, known as codon usage bias, is still controversial. In order to shed light on this point, we propose a new codon bias index, $CompAI$, that is based on the competition between cognate and near-cognate tRNAs during translation, without being tuned to the usage bias of highly expressed genes. We perform a genome-wide evaluation of codon bias for $E.coli$, comparing $CompAI$ with other widely used indices: $tAI$, $CAI$, and $Nc$. We show that $CompAI$ and $tAI$ capture similar information by being positively correlated with gene conservation, measured by ERI, and essentiality, whereas, $CAI$ and $Nc$ appear to be less sensitive to evolutionary-functional parameters. Notably, the rate of variation of $tAI$ and $CompAI$ with ERI allows to obtain sets of genes that consistently belong to specific clusters of orthologous genes (COGs). We also investigate the correlation of codon bias at the genomic level with the network features of protein-protein interactions in $E.coli$. We find that the most densely connected communities of the network share a similar level of codon bias (as measured by $CompAI$ and $tAI$). Conversely, a small difference in codon bias between two genes is, statistically, a prerequisite for the corresponding proteins to interact. Importantly, among all codon bias indices, $CompAI$ turns out to have the most coherent distribution over the communities of the interactome, pointing to the significance of competition among cognate and near-cognate tRNAs for explaining codon usage adaptation

Doctor of Philosophy

dissertationThirty four million people are infected with HIV, the causative agent of acquired immunodeficiency syndrome (AIDS), which has caused an estimated 25 million deaths in the three decades since the virus was identified. Dramatic progress has been made in understanding the viral lifecycle, enabling the development of safe and effective antiretroviral therapies that greatly reduce AIDS-associated mortality. Viral entry is mediated by the viral glycoprotein, Env. Env, composed of gp120 and gp41 subunits, adopts a transient conformation known as the prehairpin intermediate during fusion that is vulnerable to inhibition by peptides that bind the gp41 N-trimer region. Our lab has developed D-peptide inhibitors that bind to the N-trimer and prevent viral entry. The HIV lifecycle, current therapeutics, and emerging fields of HIV prevention and treatment are described in Chapter 1. Chapter 2 describes the discovery of highly potent D-peptide inhibitors of entry that bind to the N-trimer region and inhibit viral entry. These peptides, optimized by structure-guided mirror-image phage display, inhibit viral entry with nM potency. Trimerization of D-peptide inhibitors yields significant further improvement in potency due to avidity, and inhibit a broad panel of primary isolates. D-peptides are insensitive to proteolysis, a significant advantage over L-peptide inhibitors of entry

Structural aspects of translation termination on the ribosome

Translation of genetic information encoded in messenger RNAs into polypeptide sequences is carried out by ribosomes in all organisms. When a full protein is synthesized, a stop codon positioned in the ribosomal A site signals termination of translation and protein release. Translation termination depends on class I release factors. Recently, atomic-resolution crystal structures were determined for bacterial 70S ribosome termination complexes bound with release factors RF1 or RF2. In combination with recent biochemical studies, the structures resolve long-standing questions about translation termination. They bring insights into the mechanisms of recognition of all three stop codons, peptidyl-tRNA hydrolysis, and coordination of stop-codon recognition with peptidyl-tRNA hydrolysis. In this review, the structural aspects of these mechanisms are discussed