Molecular dynamics simulation exploration of unfolding and refolding of a ten-amino acid miniprotein

Steered molecular dynamics simulations are performed to explore the unfolding and refolding processes of CLN025, a 10-residue beta-hairpin. In unfolding process, when CLN025 is pulled along the termini, the force-extension curve goes back and forth between negative and positive values not long after the beginning of simulation. That is so different from what happens in other peptides, where force is positive most of the time. The abnormal phenomenon indicates that electrostatic interaction between the charged termini plays an important role in the stability of the beta-hairpin. In the refolding process, the collapse to beta-hairpin-like conformations is very fast, within only 3.6 ns, which is driven by hydrophobic interactions at the termini, as the hydrophobic cluster involves aromatic rings of Tyr1, Tyr2, Trp9, and Tyr10. Our simulations improve the understanding on the structure and function of this type of miniprotein and will be helpful to further investigate the unfolding and refolding of more complex proteins.     

Combining time‐resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin‐curcumin complex during unfolding and refolding processes

We investigated the complex interaction between bovine serum albumin (BSA) and curcumin by combining time‐resolved fluorescence and synchronous fluorescence spectroscopy. The interaction was significant and sensitive to fluorescence lifetime and synchronous fluorescence characteristics. Binding of curcumin significantly shortened the fluorescence lifetime of BSA with a bi‐molecular quenching rate constant of k_q = 3.17 × 10¹² M^‐1s^‐1. Denaturation by urea unfolded the protein molecule by quenching the fluorescence lifetime of BSA. The tyrosine synchronous fluorescence spectra were blue shifted whereas the position of tryptophan synchronous fluorescence spectra was red shifted during the unfolding process. However, denaturation of urea had little effect on the synchronous fluorescence peak of tyrosine in curcumin‐BSA complex except in the low concentration range; however, it shifted the peak to the red, indicating that curcumin shifted tryptophan moiety to a more polar environment in the unfolded state. Decreases in the time‐resolved fluorescence lifetime and curcumin‐BSA complex during unfolding were recovered during refolding of BSA by a dilution process, suggesting partial reversibility of the unfolding process for both BSA and curcumin‐BSA complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.     

Three-stage refolding/unfolding of the dual-color beta-subunit in R-phycocyanin from Polysiphonia urceolata

The conformational changes during refolding and unfolding of the dual-color beta-subunit in R-phycocyanin (R-PC) were monitored by the spectra, fluorescence anisotropy, and FRET. It was observed that both of the refolding and unfolding of the beta-subunit would undergo a three-stage conformational change, but in a reverse order. During the refolding process, at the first stage, the configuration of the tetrapyrrole chromophores transformed from the cyclohelical to the extended one, suggested by the blue-shifted spectra. At the second stage, recovery of the hydrogen-bond and hydrophobic interaction network fixed the chromophore in a more rigid configuration, suggested by a linear increase in the total fluorescence yield. At the third stage, the increase of the FRET efficiency suggested a protein-framework movement that made the two chromophores closer or/and into a more parallel orientation. The fluorescence anisotropy further confirmed the three-stage model.     

Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.     

Interactions between bovine serum albumin and gemini surfactant alkanediyl-alpha, omega-bis(dimethyldodecyl-ammonium bromide)

Interactions between bovine serum albumin (BSA) and cationic gemini surfactant alkanediyl-alpha,omega-bis(dimethyldodecyl-ammonium bromide) (12-n-12, n=3, 4, 6) in aqueous solution have been investigated by measuring fluorescence, UV-vis transmittance, dynamic lighting scattering, and circular dichroism. Compared to a traditional surfactant dodecyltrimethylammonium bromide (DTAB), 12-n-12 interacts with BSA more strongly. With increasing concentration, 12-n-12 first binds specifically onto BSA leading to the unfolding and aggregation of BSA, and the decrease in alpha-helix content; and then forms micelle-like complexes along the unfolded BSA chains. A gemini surfactant with a longer spacer has a larger effect on BSA unfolding due to a stronger hydrophobic interaction.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic <Emphasis Type="Italic">Kocuria varians</Emphasis> Alpha-Amylase

Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T_1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T_1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Refolding of chemically denatured alpha-amylase in dilution additive mode

Cyclodextrins (CDs) possess hydrophobic surfaces, which probably shield the hydrophobic surfaces of denatured proteins and prevent the direct interactions between the surfaces which are believed to be responsible for protein aggregation during refolding process. This probability was evaluated by studying the refolding process of denatured alpha-amylase in the presence and absence of alpha-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM alpha-CD in the refolding buffer, the extent of aggregation reduces by almost 90%. Spectrofluorometric analysis of the refolding intermediate(s) also indicates that the tertiary structure of the refolded alpha-amylase, in the presence of alpha-CD, is very similar to the tertiary structure of the native protein. However, this similarity was distorted upon addition of exogenous hydrophobic (aliphatic or aromatic) amino acids to the refolding buffer, meaning that the hydrophobic interactions between alpha-CD and the denatured protein play significant role in preventing aggregate formation. In addition, by weakening the extent of these hydrophobic interactions by adding polarity-reducing agent (e.g. ethylene glycol) to the refolding buffer, more aggregates were formed. In contrast, strengthening these interactions by enhancing the ionic strength of the refolding buffer made these hydrophobic interactions very strong. Therefore, alpha-CD could not depart from the protein/alpha-CD complex, as it usually does during the process of refolding. As a result, more aggregates were formed in the presence of alpha-CD compared to the corresponding control samples.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

An unfolding story of helical transmembrane proteins

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS-perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems, inverse micelles, and fragment association studies.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Understanding beta-hairpin formation by molecular dynamics simulations of unfolding

We have studied the mechanism of formation of a 16-residue beta-hairpin from the protein GB1 using molecular dynamics simulations in an aqueous environment. The analysis of unfolding trajectories at high temperatures suggests a refolding pathway consisting of several transient intermediates. The changes in the interaction energies of residues are related with the structural changes during the unfolding of the hairpin. The electrostatic energies of the residues in the turn region are found to be responsible for the transition between the folded state and the hydrophobic core state. The van der Waals interaction energies of the residues in the hydrophobic core reflect the behavior of the radius of gyration of the core region. We have examined the opposing influences of the protein-protein (PP) energy, which favors the native state, and the protein-solvent (PS) energy, which favors unfolding, in the formation of the beta-hairpin structure. It is found that the behavior of the electrostatic components of PP and PS energies reflects the structural changes associated with the loss of backbone hydrogen bonding. Relative changes in the PP and PS van der Waals interactions are related with the disruption of the hydrophobic core of a protein. The results of the simulations support the hydrophobic collapse mechanism of beta-hairpin folding.