Calcium regulates many biological processes through interaction with proteins with different conformational, dynamic, and metal binding properties. Previous studies have shown that the electrostatic environment plays a key role in calcium binding affinity. In this research, we aim to dissect the contribution of the electrostatic environment to calcium binding affinity using protein design. Many natural calcium binding proteins undergo large conformational changes upon calcium binding which hampers the study of these proteins. In addition, cooperativity between multiple calcium binding sites makes it difficult to study site-specific binding affinity. The design of a single calcium binding site into a host system eliminates the difficulties that occur in the study of calcium binding affinity. Using a computer algorithm we have rationally designed several calcium binding sites with a pentagonal bipyramidal geometry in the non-calcium dependent cell adhesion protein CD2 (CD2-D1) to better investigate the key factors that affect calcium binding affinity. The first generation proteins are all in varying electrostatic environments. The conformational and metal binding properties of each of these designed proteins were analyzed. The second generation designed protein, CD2.6D79, was designed based on criteria learned from the first generation proteins. This protein contains a novel calcium binding site with ligands all from the â-strands of the non-calcium dependent cell adhesion protein CD2. The resulting protein maintains native secondary and tertiary packing and folding properties. In addition to its selectivity for calcium over other mono and divalent metal ions, it displays strong metal binding affinities for calcium and its analogues terbium and lanthanum. Furthermore, our designed protein binds CD48, the ligand binding partner of CD2, with an affinity three-fold stronger than CD2. The electrostatic potential of the calcium binding site was modified through mutation to facilitate the study of the effect of electrostatic interactions on calcium binding affinity. Several charge distribution mutants display varying metal binding affinities based on their charge, distance to the calcium binding site, and protein stability. This study will provide insight into the key site factors that control calcium binding affinity and calcium dependent biological function.

The free energy landscape of a protein-like chain is constructed from exhaustive simulation studies using a combination of discontinuous molecular dynamics and parallel tempering methods. The protein model is a repeating sequence of four kinds of monomers, in which hydrogen bond attraction, electrostatic repulsion, and covalent bond vibrations are modeled by step, shoulder and square-well potentials, respectively. These protein-like chains exhibit a helical structure in their folded states. The model allows a natural definition of a configuration by considering which beads are bonded. In the absence of a solvent, the relative free energy of dominant structures is determined from the relative populations, and the probabilities predicted from the calculated free energies are found to be in excellent agreement with the observed probabilities at different temperatures. The free energy landscape of the protein-like chain is analyzed and confirmed to have funnel-like characteristics, confirmed by the fact that the probability of observing the most common configuration approaches unity at low enough temperatures for chains with fewer than 30 beads. The effect on the free energy landscape of an explicit square-well solvent, where the beads that can form intra-chain bonds can also form (weaker) bonds with solvent molecules while other beads are insoluble, is also examined. Simulations for chains of 15, 20 and 25 beads show that at low temperatures, the most likely structures are collapsed helical structures. The temperature at which collapsed helical structures become dominant is higher than in the absence of a solvent. Finally, the dynamics of the protein-like chain immersed in an implicit hard sphere solvent is studied using a simple model in which the implicit solvent interacts on a fast time scale with the chain beads and provides sufficient friction so that the motion of monomers is governed by the Smoluchowski equation. Using a Markovian model of the kinetics of transitions between conformations, the equilibration process from an ensemble of initially extended configurations to mainly folded configurations is investigated at low effective temperatures for a number of different chain lengths. It was observed that folding profiles appear to be single exponentials and independent of temperature at low temperatures.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

Water plays a critical role in the function and structure of biological systems. Current techniques to study biologically relevant events that span many length and time scales are limited by the prohibitive computational cost of including accurate effects from the aqueous environment. The aim of this work is to expand the reach of current molecular dynamics techniques by reducing the computational cost for achieving an accurate description of water and its effects on biomolecular systems. This work builds from the assumption that the “local” effect of water (e.g. the local orientational preferences and hydrogen bonding) can be effectively modelled considering only the atomistic detail in a very limited region. A recent adaptive resolution simulation technique (AdResS) has been developed to practically apply this idea; in this work it will be extended to systems of simple hydrophobic solutes to determine a characteristic length for which thermodynamic, structural, and dynamic properties are preserved near the solute. This characteristic length can then be used for simulation of biomolecular systems, specifically those involving protein dynamics in water. Before this can be done, current coarse grain models must be adapted to couple with a coarse grain model of water. This thesis is organized in to five chapters. The first will give an overview of water, and the current methodologies used to simulate water in biological systems. The second chapter will describe the AdResS technique and its application to simple test systems. The third chapter will show that this method can be used to accurately describe hydrophobic solutes in water. The fourth chapter describes the use of coarse grain models as a starting point for targeted search with all-atom models. The final chapter will describe attempts to couple a coarse grain model of a protein with a single-site model for water, and it’s implications for future multi-resolution studies.

Two global optimization methods are proposed in this thesis. They are the multicanonical basin hopping (MUBH) method and the basin paving (BP) method. <br /><br /> The MUBH method combines the basin hopping (BH) method, which can be used to efficiently map out an energy landscape associated with local minima, with the multicanonical Monte Carlo (MUCA) method, which encourages the system to move out of energy traps during the computation. It is found to be more efficient than the original BH method when applied to the Lennard-Jones systems containing 150-185 particles. <br /><br /> The asynchronous multicanonical basin hopping (AMUBH) method, a parallelization of the MUBH method, is also implemented using the message passing interface (MPI) to take advantage of the full usage of multiprocessors in either a homogeneous or a heterogeneous computational environment. AMUBH, MUBH and BH are used together to find the global minimum structures for Co nanoclusters with system size <em>N</em>&le;200. <br /><br /> The BP method is based on the BH method and the idea of the energy landscape paving (ELP) strategy. In comparison with the acceptance scheme of the ELP method, moving towards the low energy region is enhanced and no low energy configuration may be missed during the simulation. The applications to both the pentapeptide Met-enkephalin and the villin subdomain HP-36 locate new configurations having energies lower than those determined previously. <br /><br /> The MUBH, BP and BH methods are further employed to search for the global minimum structures of several proteins/peptides using the ECEPP/2 and ECEPP/3 force fields. These two force fields may produce global minima with different structures. The present study indicates that the global minimum determination from ECEPP/3 prefers helical structures. Also discussed in this thesis is the effect of the environment on the formation of beta hairpins.

<p>As of late 2009, the Protein Data Bank (PDB) has grown to contain over 70,000 models. This recent increase in the amount of structural data allows for more extensive explication of the governing principles of macromolecular folding and association to complement traditional studies focused on a single molecule or complex. PDB-wide characterization of structural features yields insights that are useful in prediction and validation of the 3D structure of macromolecules and their complexes. Here, these insights lead to a deeper understanding of protein--protein interfaces, full-atom critical assessment of increasingly more accurate structure predictions, a better defined library of RNA backbone conformers for validation and building 3D models, and knowledge-based automatic correction of errors in protein sidechain rotamers. </p><p>My study of protein--protein interfaces identifies amino acid pairing preferences in a set of 146 transient interfaces. Using a geometric interface surface definition devoid of arbitrary cutoffs common to previous studies of interface composition, I calculate inter- and intrachain amino acid pairing preferences. As expected, salt-bridges and hydrophobic patches are prevalent, but likelihood correction of observed pairing frequencies reveals some surprising pairing preferences, such as Cys-His interchain pairs and Met-Met intrachain pairs. To complement my statistical observations, I introduce a 2D visualization of the 3D interface surface that can display a variety of interface characteristics, including residue type, atomic distance and backbone/sidechain composition. </p><p>My study of protein interiors finds that 3D structure prediction from sequence (as part of the CASP experiment) is very close to full-atom accuracy. Validation of structure prediction should therefore consider all atom positions instead of the traditional Calpha-only evaluation. I introduce six new full-model quality criteria to assess the accuracy of CASP predictions, which demonstrate that groups who use structural knowledge culled from the PDB to inform their prediction protocols produce the most accurate results. </p><p>My study of RNA backbone introduces a set of rotamer-like "suite" conformers. Initially hand-identified by the Richardson laboratory, these 7D conformers represent backbone segments that are found to be genuine and favorable. X-ray crystallographers can use backbone conformers for model building in often poor backbone density and in validation after refinement. Increasing amounts of high quality RNA data allow for improved conformer identification, but also complicate hand-curation. I demonstrate that affinity propagation successfully differentiates between two related but distinct suite conformers, and is a useful tool for automated conformer clustering. </p><p>My study of protein sidechain rotamers in X-ray structures identifies a class of systematic errors that results in sidechains misfit by approximately 180 degrees. I introduce Autofix, a method for automated detection and correction of such errors. Autofix corrects over 40% of errors for Leu, Thr, and Val residues, and a significant number of Arg residues. On average, Autofix made four corrections per PDB file in 945 X-ray structures. Autofix will be implemented into MolProbity and PHENIX for easy integration into X-ray crystallography workflows.</p> / Dissertation

CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.

We used the combination of molecular dynamics and Monte Carlo method to investigate protein folding problems. The environments of proteins are very big, and often very time-consuming. If simulations are based on traditional methods of molecular simulations, it will cost very long time to accomplish the simulation. We use a special designed method, in which the molecular dynamics is used for determining the soft part of protein, and use Monte Carlo method to move and rotate the bonds of proteins. By removing a lot impossible movements in traditional Monte Carlo method, we shorten simulation time and simulate protein folding process effectively. In this work, we used GBSA solvent model, AMBER force field, and semi-local movements to accelerate the simulations. We obtained good result by this simulation method of a small peptide 1L2Y.

A thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.

In the cell, as in in vitro, the final conformation of a protein is determined by it's amino acid sequence (1). Some isolated proteins can be denatured and refolded in vitro in absence of extrinsic factors. However, in order to fold in the cell, the newly synthesized polypeptide chain has to negotiate an environment far more complex than that faced by the unfolded chain in vitro. Cells have evolved proteins called “chaperones” to assist folding and assembly of polypeptides (2). Thus, the linear sequence of a protein not only contains information that specifies the final three-dimensional functional form, but also recognition motifs, which can be recognized by the cellular folding machinery. The work reported in this thesis is aimed at understanding some aspects of recognition of target substrates by the cytosolic chaperone, SecB, which forms part of the protein translocation machinery in E. coli. The sec pathway is involved in both translocation of precursor proteins across and the insertion of integral membrane proteins into the cytoplasmic membrane (3). Chapter one discusses some general aspects of protein folding and briefly describes chaperone systems, which have been extensively characterized in literature. Chapter two discusses the effect of chaperone SecB on the refolding pathway of a model substrate protein barstar, whose folding pathway has been extensively characterized (4,5). The effect of SecB on the refolding kinetics of the small protein barstar (wild type) and fluorescein labeled C82A (single Cys mutant) in 1 M guanidine hydrochloride at pH 7.0 at 25 °C has been investigated using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to late near-native intermediate (s) along the folding pathway. ESR studies and fluorescence anisotropy measurements show that SecB forms stable complexes with the near-native intermediate (s). For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. Steady state polarization measurements indicated the presence of stable complexes of barstar bound to SecB. Studies on the spin labeled C82A show an immobilization of the spin label adduct at the 40th position of barstar, suggesting that the binding of SecB to barstar occurs in that region. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models (6). Chapter three discusses the energetics of substrate:SecB interactions using the following model protein substrates: unfolded RNase A, BPTI, partially folded disulfide intermediates of alpha-lactalbumin,. The thermodynamics of binding of unfolded polypeptides to the chaperone SecB were investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29 and -0.41 kcal mol-1 K-1 respectively and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft (7). Chapter four discusses the thermodynamics of unfolding to gain insights into the mechanism of assembly and stability of the tetrameric structure. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. The value of ACP obtained was 10.7 ± 0.7 kcal mol-1 K-1, which is amongst the highest measured for a multimeric protein. At 298 K, pH 7.4. the AG°U for the SecB tetramer is 27.9 ± 2 kcal mol-1. Denaturant mediated unfolding of SecB was found to be irreversible. The reactivity of the 4 solvent exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four Cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well folded and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity (8). Chapter five discusses the bound state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI), as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58 residue protein and contains 3 disulfide groups between residues 5 and 55, 14 and 38, and 30 and 51. Single disulfide mutants of BPTI were reduced and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four, solvent accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The ESR data suggest that these cysteine residues are in close proximity when no substrate protein is bound, but move away from each other when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein. Chapter six discusses the mechanism of dissaggregation of a model peptide aggregate by chaperone SecB. The Hspl04, Hsp70 and Hsp40 chaperone system are capable of dissociating aggregated state(s) of substrate proteins, though little is known of the mechanism of the process. The interaction of the B chain of insulin with chaperone SecB was investigated using light scattering, pyrene excimer fluorescence and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B chain of insulin. We show that SecB is capable of dissociating soluble B chain aggregate as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B chain aggregate by SecB has also been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B chains, rather it binds the small population of free B chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate towards the individual B chains. Thus SecB can rescue aggregated, partially folded /misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B chain to chaperone SecB. The data suggests that the B chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate (9).