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.

The Major Intrinsic Proteins are represented in all forms of life; plants, animals, bacteria and recently archaebacteria have all been shown to express at least one member of this superfamily of integral membrane proteins. We have overexpressed the E. coli aquaglyceroporin, glycerol facilitator (GlpF), to use as a model for studying membrane protein structure, folding and stability. Understanding membrane protein folding, stability, and dynamics is required for a molecular explanation of membrane protein function and for the development of interventions for the hundreds of membrane protein folding diseases. X-ray analysis of GlpF crystals shows that the protein exits as a tetramer in the crystallized state [1]. However, preparations of stable aqueous detergent solutions of GlpF in its native oligomeric state have been difficult to make; the protein readily unfolds and forms non-specific aggregates in many detergents. Here, I report the study of the structure and stability of the glycerol facilitator in several detergent solutions by blue native and sodium dodecyl sulphate polyacrylamide gel electrophoresis, circular dichroism, and fluorescence. For the first time, stable protein tetramers were prepared in two different detergent solutions (dodecyl maltoside (DDM) and lyso-myristoyl phosphatidylcholine (LMPC)) at neutral pH. Thermal unfolding experiments show that the protein is slightly more stable in LMPC than in DDM and that the thermal stability of the helical core at 95oC is slightly greater in the former detergent. In addition, tertiary structure unfolds before quaternary and secondary structures in LMPC whereas unfolding is more cooperative in DDM. The high stability of the protein is also evident from the unfolding half-life of 8 days in 8 M urea suggesting that hydrophobic interactions contribute to the stability. The GlpF tetramers are less resistant to acidic conditions; LMPC-solubilized GlpF shows loss of tertiary and quaternary structure by pH 6, while in DDM the tertiary structure is lost by pH 5, however the tetramer remains mostly intact at pH 4. The implications of thermal and chemical stress on the stability of the detergent-solubilized protein and its in vivo folding are discussed.

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.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

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;