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Orientador: Sergio Daishi Sasaki / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2013

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).

The oxidative folding pathway of the disulfide containing protein bovine pancreatic trypsin inhibitor (BPTI) was one of the first to be elucidated and has served as a basis for understanding the folding pathways of other proteins. During the oxidative folding of reduced BPTI, two intermediates (N' and N*) accumulate in significant amounts and act as kinetic traps. Both N' and N* bury their two remaining free thiols in their hydrophobic cores, which inhibits further oxidation. Historically, the rate limiting step was considered to be the intramolecular rearrangements of N' and N* to another intermediate with two free thiols, NSH. The two free thiols in NSH are solvent-exposed and easily oxidized to a disulfide, producing native protein (N). Nevertheless, our research using reduced BPTI indicated that the folding rate of N* to N was proportional to the concentration of added glutathione disulfide (GSSG), inconsistent with the slow intramolecular rearrangement of N* to NSH. To confirm our initial results, the intermediate N* was purified and refolded in the presence of GSSG. The conversion of N* to N was dependent upon the disulfide concentration and singly mixed disulfide N*(SG) was observed during folding. These results emphasize that the folding of N* can proceed via a growth type pathway, direct oxidation of the two remaining thiols in N* by an exogenous small molecule disulfide, such as GSSG, to form N. Folding of reduced BPTI via N* was performed under changing concentrations of GSSG and GSH as a function of time. The folding was improved dramatically in terms of rate and yield. Aromatic disulfides and thiols have been demonstrated to improve the folding efficiency of disulfide containing proteins including ribonuclease A (RNase A) and lysozyme. Herein, N* and N' were refolded in the presence of aromatic disulfides. Folding of the two kinetic traps with aromatic disulfides indicated that folding proceed via a growth type pathway. The singly and doubly mixed disulfide intermediates were observed during most folding reactions. The oxidative folding of reduced BPTI with aromatic disulfides and thiols were also investigated. Reduced BPTI can be folded to disulfide intermediates rapidly.

Almost all therapeutic proteins contain disulfide bonds to stabilize their native structure. Recombinant DNA technology enables many therapeutic proteins to be produced in bacteria, but the expression of native proteins is not always efficient due to the limited ability of bacteria to form disulfide bonds in vivo. It is often necessary to employ in vitro oxidative folding process to form the native disulfide bonds to obtain the native structure of disulfide-containing proteins. Aromatic disulfides are small molecules designed to match some of the physical properties of the active site of protein disulfide isomerase (PDI), which catalyzes the folding process of disulfide-containing proteins in eukaryotes. Three aromatic thiols with varying charges, PA, SA and QAS thiol, were used to fold reduced BPTI in vitro. Bovine pancreatic trypsin inhibitor (BPTI) is positively charged (pI = 10.5) at pH 7.3, and we hypothesized that mixed disulfide intermediates formed between BPTI and negatively charged small molecule thiols were more likely to precipitate due to their minimized net charge. Protein precipitation was observed during folding with negatively charged thiols, PA and SA, but not positively charged thiol QAS. At the folding pH of 7.3, almost 90% of native BPTI was produced in 2 h with the conditions of 0.25 mM QAS disulfide and 10 mM QAS thiol. Only 25% of native BPTI was produced in 2 h with the best conditions for glutathione and glutathione disulfide. Aromatic thiols with an elongated alkyl group on the aromatic ring, butyl, hexyl and octyl thiol, were hypothesized to increased interactions with the hydrophobic core of disulfide-containing proteins during folding, allowing more facile access to buried disulfide bonds. However, the longer the hydrocarbon chain, the more likely protein precipitation was to occur. About 90% native BPTI was formed in 1 h with 0.25 mM hexyl disulfide and 10 mM hexyl thiol. A method using capillary electrophoresis (CE) to analysis the oxidative folding process of reduced BPTI with small molecule thiols and disulfides was also developed. Folding of reduced BPTI with QAS disulfide was analyzed using CE in a shorter run time. The consumption of protein samples and solvent solutions was minimized.

Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide. Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein. Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.