Toward a Database of Geometric Interrelationships of Protein Secondary Structure Elements for De Novo Protein Design, Prediction and Analysis

Computational methods of analyzing, simulating, and modeling proteins are essential towards understanding protein structure and its interactions. Computational methods are easier as not all protein structures can be determined experimentally due to the inherent difficultly of working with some proteins. In order to predict, design, analyze, simulate or model a protein, data from experimentally determined proteins such as those located in the repository of the Protein Data Bank (PDB) are essential. The assumption here is that we can use pieces of known proteins to piece together a new protein hence, de novo protein design. The analysis of the geometric relationships between secondary structure elements in proteins can be extremely useful to protein prediction, analysis, and de novo design. This thesis project involves creating a database of protein secondary structure elements and geometric information for rapid protein assembly, de novo protein design, prediction and analysis

Toward a Database of Geometric Interrelationships of Protein Secondary Structure Elements for De Novo Protein Design, Prediction and Analysis

Computational methods of analyzing, simulating, and modeling proteins are essential towards understanding protein structure and its interactions. Computational methods are easier as not all protein structures can be determined experimentally due to the inherent difficultly of working with some proteins. In order to predict, design, analyze, simulate or model a protein, data from experimentally determined proteins such as those located in the repository of the Protein Data Bank (PDB) are essential. The assumption here is that we can use pieces of known proteins to piece together a new protein hence, de novo protein design. The analysis of the geometric relationships between secondary structure elements in proteins can be extremely useful to protein prediction, analysis, and de novo design. This thesis project involves creating a database of protein secondary structure elements and geometric information for rapid protein assembly, de novo protein design, prediction and analysis

Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane

Structural dynamics and membrane interaction of the chloride intracellular channel protein, CLIC1

ABSTRACT The Chloride Intracellular Channel (CLIC) proteins are a family of amphitropic proteins that can convert from soluble to integral membrane forms. CLIC1 is a member of this family that functions as a chloride channel in the plasma and nuclear membranes of cells. Although high-resolution structural data exists for the soluble form of monomeric CLIC1, not much is known about the integral membrane forms’ structure. The exact mechanism and signals involved in the conversion of the soluble form to membrane-inserted form are also not clear. Studies were undertaken in the absence and presence of membrane models. Analysis of the structure and stability of CLIC1 in the absence of membrane investigated the effect of possible signals or triggers that may play a crucial role in the conversion of the soluble form to integral membrane form. Exposing CLIC1 to oxidizing conditions results in the formation of a dimeric form. The CLIC1 dimer was found to be less stable than the monomeric form based on unfolding kinetic studies. The stability of the dimer was also less influenced by salt concentration, compared with the monomer. The effect of pH on the structure of CLIC1 is of physiological relevance since the movement of soluble CLIC1 in the cytoplasm or nucleoplasm toward the membrane will involve the protein being exposed to a lower pH micro-environment. Hydrogen exchange mass spectrometry was used to study the structural dynamics of CLIC1 at pH 7.0 and pH 5.5. At neutral pH, domain II is more stable than the more flexible thioredoxin domain I. The thioredoxin-fold therefore is more likely to unfold and rearrange to insert into membranes. Because of the high stability of domain II this region is probably where the folding nucleus of the protein is. At pH 5.5 it was found that the a1, a3 and a6 helices, which are spatially adjacent to one another across the domain interface, were destabilized. This destabilization may be the trigger for CLIC1 to unfold and rearrange into a membrane insertion-competent form. The role of the primary sequence and unique three-dimensional structure of CLIC1 in membrane insertion was investigated in a bioinformatics-based study that looked at conserved residue features such as hydropathy and charge. Hidden helical propensities and Ncapping motifs in the a1-b2 region were found, which may have important implications for locating putative transmembrane regions. Analysis of the structure and thermodynamics of CLIC1 interacting with membranes investigated changes in secondary structure, tertiary structure, hydrodynamic volume and thermodynamics when CLIC1 is exposed to membrane-mimicking models. The effect of a variety of conditions such as pH and redox, cysteine-modifiying agents (NEM), ligands (GSH), and inhibitors (IAA) on CLIC1 membrane interaction were studied. It was found that CLIC1 interacted with membranes more favourably at lower pH and that NEM completely inhibited CLIC1 interaction with micelles

Development of genetic algorithm for optimisation of predicted membrane protein structures

Due to the inherent problems with their structural elucidation in the laboratory, the computational prediction of membrane protein structure is an essential step toward understanding the function of these leading targets for drug discovery. In this work, the development of a genetic algorithm technique is described that is able to generate predictive 3D structures of membrane proteins in an ab initio fashion that possess high stability and similarity to the native structure. This is accomplished through optimisation of the distances between TM regions and the end-on rotation of each TM helix. The starting point for the genetic algorithm is from the model of general TM region arrangement predicted using the TMRelate program. From these approximate starting coordinates, the TMBuilder program is used to generate the helical backbone 3D coordinates. The amino acid side chains are constructed using the MaxSprout algorithm. The genetic algorithm is designed to represent a TM protein structure by encoding each alpha carbon atom starting position, the starting atom of the initial residue of each helix, and operates by manipulating these starting positions. To evaluate each predicted structure, the SwissPDBViewer software (incorporating the GROMOS force field software) is employed to calculate the free potential energy. For the first time, a GA has been successfully applied to the problem of predicting membrane protein structure. Comparison between newly predicted structures (tests) and the native structure (control) indicate that the developed GA approach represents an efficient and fast method for refinement of predicted TM protein structures. Further enhancement of the performance of the GA allows the TMGA system to generate predictive structures with comparable energetic stability and reasonable structural similarity to the native structure

Computational Study of the Human Dystrophin Repeats: Interaction Properties and Molecular Dynamics

Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy

Molecular dynamics study of melatonin binding to homology modelled Mel1a G-protein coupled receptor

The Mel1a G-coupled Protein receptor (GPCR) was modelled using the I-Tasser online web service. All-atom molecular dynamics was used to improve the structure. The primary ligand melatonin was docked to the structure post molecular dynamics and structurally aligned to the X-ray crystallographic structures of the β2 adrenergic and rhodopsin GPCR’s, of the same family of proteins. A second set of all-atom molecular dynamics was undertaken with melatonin in the proposed active site which was parameterized ab initio in Gaussian16 to note any key conformational changes due to binding. The Mel1a GPCR becomes depolarized as a result of binding in the proposed active site by melatonin, based on Van der Waal interaction with amino acid residues on the extracellular side of the membrane (Ser176, Cys177, Tyr281 and Ser103)

Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We have performed extensive molecular dynamics simulations of these five PrP mutants and compared their dynamics and conformations to wild-type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases