The refolding of recombinant human liver methylmalonyl-CoA mutase from inclusion bodies produced in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as pan of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the purification of recombinant E. coli chaperonins GroES and GroEL from the E. coli strain DH1/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5'adenosylcobalamin- agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~3.11 units/mg

The aggregation of cytochrome C may be linked to its flexibility during refolding

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as -helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like -helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of HL from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate 0.1 s1, which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle

Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region

The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Roman, Ernesto Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed

Folding Under Inequilibrium Conditions as a Possible Reason for Partial Irreversibility of Heat-Denatured Proteins: Computer Simulation Study

Using computer simulations we have studied possible effects of heating and cooling at different scan rates on unfolding and refolding of macromolecules. We have shown that even the simplest two-state reversible transition can behave irreversibly when an unfavorable combination of cooling rate, relaxation time and activation energy of refolding occurs. On the basis of this finding we suppose that apparent irreversibility of some proteins denatured by heat may result from slow relaxation on cooling rather than thermodynamic instability and/or irreversible alterations of the polypeptide chain. Using this kinetic reversible two-state model, we estimated the effects of the scan rate and kinetic parameters of the macromolecule on its unfolding–refolding process. A few recommendations are suggested on how to reach maximal possible recovery after denaturation if refolding appears to be under kinetic control

Refolding dynamics of stretched biopolymers upon force quench

Single molecule force spectroscopy methods can be used to generate folding trajectories of biopolymers from arbitrary regions of the folding landscape. We illustrate the complexity of the folding kinetics and generic aspects of the collapse of RNA and proteins upon force quench, using simulations of an RNA hairpin and theory based on the de Gennes model for homopolymer collapse. The folding time, $\tau_F$, depends asymmetrically on $\delta f_S = f_S - f_m$ and $\delta f_Q = f_m - f_Q$ where $f_S$ ($f_Q$) is the stretch (quench) force, and $f_m$ is the transition mid-force of the RNA hairpin. In accord with experiments, the relaxation kinetics of the molecular extension, $R(t)$, occurs in three stages: a rapid initial decrease in the extension is followed by a plateau, and finally an abrupt reduction in $R(t)$ that occurs as the native state is approached. The duration of the plateau increases as $\lambda =\tau_Q/\tau_F$ decreases (where $\tau_Q$ is the time in which the force is reduced from $f_S$ to $f_Q$). Variations in the mechanisms of force quench relaxation as $\lambda$ is altered are reflected in the experimentally measurable time-dependent entropy, which is computed directly from the folding trajectories. An analytical solution of the de Gennes model under tension reproduces the multistage stage kinetics in $R(t)$. The prediction that the initial stages of collapse should also be a generic feature of polymers is validated by simulation of the kinetics of toroid (globule) formation in semiflexible (flexible) homopolymers in poor solvents upon quenching the force from a fully stretched state. Our findings give a unified explanation for multiple disparate experimental observations of protein folding.Comment: 31 pages 11 figure

Mapping the Cytochrome C Folding Landscape

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S. cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a timescale regulated by heme ligation events (~300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure. Nonnative states of proteins are involved in a variety of cellular processes, including translocation of proteins across membranes and formation of amyloid fibrils. Probes that report on the structural heterogeneity of a polypeptide ensemble could resolve ambiguities in the classification of these states. We have shown that added anions shift the equilibrium between the compact and extended polypeptide structures that are present during refolding of Saccaromyces cerevisiae iso-1 cytochrome c. Specifically, at high salt concentrations (>= 700 mM), all the polypeptides are compact with a mean C-terminal fluorophore-heme separation quite close to that in the native protein (25 A). Addition of chemical detaturants, on the other hand, tends to shift the equilibrium towards unfolded structures. Folding of modified Fe(II) cyt c was probed by fluorescence in presence of imidazole with NADH as photochemical sensitizer. At very high imidazole concentrations (400 mM), protein was still found to fold but the rate that coincides with Met80 ligation was slowed down significantly. Reductive flash-quench/scavenge experiments, in which ascorbic acid was used to scavenge MeODMAA+, were shown to keep ferrocyt c reduced for up to 500 ms. Electron injection into unfolded modified yeast Fe(III)cyt c was fast in comparison to injection using NADH as photochemical sensitizer. The overall electron transfer process was reversible. This photoreduction system could be used to trigger folding of Fe(II) cyt c to monitor the changes in dansyl fluorescence intensity on ms time scales.</p