Members of the Sco protein family have been proposed to function in the assembly of cytochrome c oxidase in the respiratory chain of all aerobic life forms. The Sco protein in Bacillus subtilis, BsSco, is characterized for its folding/unfolding behavior in the presence or absence of Cu(II) in this study. The folding/unfolding of apo-BsSco is investigated by CD and fluorescence spectroscopies. BsSco follows an apparent two-state mechanism in both folding and unfolding processes. The two apo forms of BsSco, reduced and oxidized, exhibit similar equilibrium stabilities suggesting that the formation of an intramolecular disulfide in oxidized apo-BsSco does not add to BsSco’s overall stability. In contrast, Cu(II) binding to reduced apo-BsSco results in extreme stabilization and resistance to unfolding in urea. However, when Cu(II) is present with unfolded, reduced apo-BsSco, the protein is rapidly oxidized. Another widely used denaturant, GdnHCl, is able to unfold Cu(II)-BsSco by allowing the loss of Cu(II) from the metal/protein complex. When the presence of Cu(II)-BsSco complex and the protein’s folded state are monitored simultaneously, the unfolding of Cu(II)-bound BsSco occurs coincidently with Cu(II) dissociation. We suggest that the loss of Cu(II) binding and the loss of BsSco’s native conformation are coincident, which leads to the conclusion that Cu(II)-BsSco does not unfold until it forfeits Cu(II). The kinetics of folding/unfolding of reduced, oxidized and Cu(II) bound BsSco are explored by stopped-flow fluorescence spectroscopy. The rate constants at which the two apo forms of BsSco fold and unfold are measured and plotted versus denaturant concentration. Reduced and oxidized forms of apo-BsSco are similar in folding and unfolding kinetics. Cu(II)-involved refolding kinetics of BsSco show that Cu(II) is able to accelerate the rate of refolding. However, the involvement of Cu(II) in the refolding process results in two competing processes: oxidation and Cu(II) binding. Which process predominates depends on the refolding rate which further depends on the denaturant concentration. This study has provided direct evidence for metal-involved stabilization of BsSco which is beneficial to efficiently fulfill its copper trafficking duty in a cellular environment. / Thesis (Master, Biochemistry) -- Queen's University, 2010-12-17 17:24:09.598

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-09-30 12:32:41.379

No description available.

The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fumarase from pig heart, the NAD+ -dependent isocitrate dehydrogenase from yeast, the citrate synthases from pig hean, Acinetobacter anitratum and Thermoplasma acidophilum and the chaperone protein GroEL from Escherichia coli. In each case the unfolding by guanidinium chloride (GdnHCI) was monitored by enzyme activity (to detect possible perturbations at the active site), protein fluorescence (to detect changes in tertiary structure) and far U.v. circular dichroism (to detect changes in protein secondary structure). In general the losses in secondary and tertiary structure were found to run broadly in parallel, whereas the enzyme activity was lost at much lower concentrations of GdnHCl. This sensitivity to mild, denaturing conditions may reflect the greater flexibility of the active site compared with the molecule as a whole. Interestingly) the bacterial citrate synthases were activated in the presence of low concentrations of GdnHCl. Following denaturation) refolding was initiated by lowering the concentration of GdnHCI by dilution or dialysis. Only the dimeric citrate synthases (from pig heart and Thermoplasma acidophilum) could be reactivated to a moderate extent using the dilution procedure; less than 5% reactivation was observed for the other enzymes. In the cases of fumarase, NAD+ -dependent isocitrate dehydrogenase and the dimeric citrate synthases the degrees of reactivation following dialysis were significantly greater (approximately 50-75% of the native enzymes) than those obtained following the dilution procedure. Factors such as protein concentration and the inclusion of dithiothreitol in the dialysis or dilution buffer were found to influence significantly the extent of reactivation. The greater yield of reactivation of unfolded protein using the dialysis procedure probably reflects the ability of the enzyme to make the correct structural adjustments between intermediates when the concentration of GdnHCI is lowered gradually. In the case of Thermoplasma acidophilum the recovery of citrate synthase activity was much greater at 20 ·C than at 55 ·C (the optimal temperature for growth of this organism). This has implications for the folding process in vivo under the extreme growth conditions of thermophiles and possibly other extremophiles. The hexameric citrate synthase from Acinetobacter anitratum and the tetradecameric chaperonin, GroEL could not be reactivated following denaturation. Far u.v. circular dichroism measurements on GroEL indicated that the native secondary structure of this protein was regained to a large extent. In vivo a number of the proteins studied (fumarase and citrate synthase from pig hean and yeast NAD+ -dependent isocitrate dehydrogenase)are translocated into mitochondria as precursors in a non-native state prior to processing, folding and assembly. The lack of complete refolding of the proteins studied in this work points to the existence of specialised mechanisms in vivo to promote efficient folding. Chaperone proteins have been implicated in the assistance of protein folding in vivo. Intriguingly. the studies on the inefficient refolding of the chaperonin GroEL support the proposal that this protein may fold in vivo by way of a "self chaperoning" mechanism.

The four cyanogen bromide fragments of yeast 3-phosphoglycerate kinase (PGK) have been isolated and characterised. After digestion with proteolytic enzymes and specific cleavage reagents, the resuting peptides were purified by various methods and sequenced using the manual dansyl-Edman technique and the Beckman 890C liquid phase sequencer. The entire sequence of yeast PGK (415 residues) has been determined using a combination of amino acid sequence data and nucleotide sequence data. Nucleotide sequence data were supplied by Dr. A. Kingsman, University of Oxford. The yeast PGK sequence data have been fitted tothe 2.S electron density map and the nucleotide binding site has been fully characterised. The fitting of sequence data to the electron density map permitted identification of additional electron density which is probably attributable to the triose phosphate substrate. This binding site has also been characterised. The construction of the 1g:1cm model of yeast PGK permitted interpretation of chemical modification, NMR, hydrodynamic and kinetic data from a structural point of veiw, thereby allowing a catalytic mechanism to be proposed. This mechanism involves a major conformational change, triggered by the breaking of a salt-bridge between glutamate 190 and histidine 388 concommittant with the formation of the ternary enzyme-substrates complex. The conformational change brings the two substrates into close proximity, thereby permitting the in-line, direct, associative,phosphoryl transfer reaction to take place. The hydrodynamic properties of yeast PGK were examined in order to determine conditions under which PGK adopted its closed , catalytically active conformation. The solubility of yeast PGK in organic solvents commonly used as crystallising media was examined and experiments performed which were designed to crystallise a) the closed conformation of yeast PGK, and b) the substrate-free form of yeast PGK. No crystals have yet been observed in these experiments.

Although integral membrane proteins represent nearly a quarter of the genes present in both prokaryotes and eukaryotes, progress in this area of research is often hindered due to the nature of their hydrophobic environment. Elucidating the folding pathway of these proteins is essential to understand many membrane mediated biological processes such as signal transduction, ion transport and chemotaxis. The wealth of structural and genetic information on bacteriorhodpsin renders it an ideal model system for the study of membrane proteins. Detailed studies however, necessitate efficient methods for its overexpression and purification. Previous expression systems have reported difficulty in obtaining good yields and simple purification procedures. This thesis investigates a variety of alternative expression and purification systems for the bacterio-opsin gene in Escherichia coli. With sufficient protein, site directed mutagenesis is performed to mutate three proline residues present in the membranous region of bacteriorhodopsin to alanine. The folding kinetics of these mutants is investigated using stopped flow fluorimetry to determine whether proline isomerisation is responsible for a slow step in the folding pathway of bacteriorhodopsin. Comparison of the results with those of the folding kinetics of wild type showed proline isomerisation not to be responsible for the slow step in the pathway. More recent studies have suggested that the slow step may be due to refolding conditions and lateral pressure the lipids impose upon the protein as well as pH. Separate structural studies using mass spectrometry aimed to study the rates of isotopic exchange of amide and side chain protons in bacteriorhodopsin. Low resolution results obtained using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) prompted the investigation of electrospray ionisation mass spectrometry (ESI-MS). Techniques for sample preparation were optimised by investigating a variety of solvent systems and initial deuteration experiments performed.

No description available.

The lumen of the endoplasmic reticulum (ER) provides a specialized environment to assure the folding and oligomerization of secretory proteins to their native conformations. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a biosensor in the ER that detects the folding state of glycoproteins. UGGT-catalyzed monoglucosylation of incompletely folded glycoproteins leads to their continued retention in the ER through their association with the lectins calnexin and calreticulin for further folding or for degradation. Purified recombinant UGGT from rat liver and glycoprotein substrates from a mutant strain of Saccharomyces cerevisiae were used in an in vitro system to examine the peptide components recognized by UGGT in unfolded glycoproteins and glycopeptides. Mass spectrometry was used to measure and quantitate the levels of glucose incorporation into these substrates that was directly related to their level of recognition by UGGT. To assess the capacity of UGGT for sensing non-native structures in glycoprotein substrates, Exo-1,3-beta-glucanase (beta-Glc) from S. cerevisiae was crystallized and its structure determined. A mutagenesis strategy was used to mutate solvent-exposed residues to yield the beta-Glc F280S point mutant that retained enzymatic activity while still being recognized by UGGT. These data suggest that UGGT recognizes solvent-exposed hydrophobic patches in the primary and tertiary structure of glycoproteins even in near-native conformations.

Oxidative protein folding within the ER depends on the enzymatic action of numerous chaperones and oxidoreductases. In addition, this process requires the influx of metabolites and energy, including FAD (flavin adenine dinucleotide) and molecular oxygen. Secretory proteins and proteins destined to the secretory pathway need to undergo this process in order to obtain stability and full functionality. Since secretory proteins that fail to fully fold are eliminated by degradation, the process of ER oxidative protein folding is part of a group of ER-associated mechanisms commonly referred to as ER quality control. Interestingly, the proteins that mediate ER quality control can be found in a variety of diverse subdomains of the ER. We have found that the ER-oxidoreductase Ero1 is located on the mitochondria-associated-membrane, the MAM. This specialized subdomain of the ER has been shown to be crucial for a number of processes such as the synthesis of phospholipids as well as calcium-channelling between the ER and mitochondria. The goal of this thesis was to identify possible retention mechanisms and motifs of Ero1 to the MAM.

Thesis (Ph.D.)--Tufts University, 2001. / Adviser: David L. Weaver. Submitted to the Dept. of Physics. Includes bibliographical references (leaves 148-149). Access restricted to members of the Tufts University community. Also available via the World Wide Web;