Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin

Extensive measurements and analysis of thermodynamic stability and kinetics of urea-induced unfolding and folding of hisactophilin are reported for 5-50 degrees C, at pH 6.7. Under these conditions hisactophilin has moderate thermodynamic stability, and equilibrium and kinetic data are well fit by a two-state transition between the native and the denatured states. Equilibrium and kinetic m values decrease with increasing temperature, and decrease with increasing denaturant concentration. The betaF values at different temperatures and urea concentrations are quite constant, however, at about 0.7. This suggests that the transition state for hisactophilin unfolding is native-like and changes little with changing solution conditions, consistent with a narrow free energy profile for the transition state. The activation enthalpy and entropy of unfolding are unusually low for hisactophilin, as is also the case for the corresponding equilibrium parameters. Conventional Arrhenius and Eyring plots for both folding and unfolding are markedly non-linear, but these plots become linear for constant DeltaG/T contours. The Gibbs free energy changes for structural changes in hisactophilin have a non-linear denaturant dependence that is comparable to non-linearities observed for many other proteins. These non-linearities can be fit for many proteins using a variation of the Tanford model, incorporating empirical quadratic denaturant dependencies for Gibbs free energies of transfer of amino acid constituents from water to urea, and changes in fractional solvent accessible surface area of protein constituents based on the known protein structures. Noteworthy exceptions that are not well fit include amyloidogenic proteins and large proteins, which may form intermediates. The model is easily implemented and should be widely applicable to analysis of urea-induced structural transitions in proteins.     

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pK_a values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.     

Tracking unfolding and refolding of single GFPmut2 molecules

The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.     

How do chemical denaturants affect the mechanical folding and unfolding of proteins?

We present the first single-molecule atomic force microscopy study on the effect of chemical denaturants on the mechanical folding/unfolding kinetics of a small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus). Upon increasing the concentration of the chemical denaturant guanidinium chloride (GdmCl), we observed a systematic decrease in the mechanical stability of GB1, indicating the softening effect of the chemical denaturant on the mechanical stability of proteins. This mechanical softening effect originates from the reduced free-energy barrier between the folded state and the unfolding transition state, which decreases linearly as a function of the denaturant concentration. Chemical denaturants, however, do not alter the mechanical unfolding pathway or shift the position of the transition state for mechanical unfolding. We also found that the folding rate constant of GB1 is slowed down by GdmCl in mechanical folding experiments. By combining the mechanical folding/unfolding kinetics of GB1 in GdmCl solution, we developed the “mechanical chevron plot” as a general tool to understand how chemical denaturants influence the mechanical folding/unfolding kinetics and free-energy diagram in a quantitative fashion. This study demonstrates great potential in combining chemical denaturation with single-molecule atomic force microscopy techniques to reveal invaluable information on the energy landscape underlying protein folding/unfolding reactions.     

Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.     

Co-translational domain folding as the structural basis for the rapid de novo folding of firefly luciferase.

The 62 kDa protein firefly luciferase folds very rapidly upon translation on eukaryotic ribosomes. In contrast, the chaperone-mediated refolding of chemically denatured luciferase occurs with significantly slower kinetics. Here we investigate the structural basis for this difference in folding kinetics. We find that an N-terminal domain of luciferase (residues 1-190) folds co-translationally, followed by rapid formation of native protein upon release of the full-length polypeptide from the ribosome. In contrast sequential domain formation is not observed during in vitro refolding. Discrete unfolding steps, corresponding to domain unfolding, are however observed when the native protein is exposed to increasing concentrations of denaturant. Thus, the co-translational folding reaction bears more similarities to the unfolding reaction than to refolding from denaturant. We propose that co-translational domain formation avoids intramolecular misfolding and may be critical in the folding of multidomain proteins.     

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.     

Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

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.     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.     

Alternatively folded states of an immunoglobulin

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Evidence of discrete substates and unfolding pathways in green fluorescent protein

We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules' switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels

Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.