A stable, molten-globule-like cytochrome c

Expression of cytochrome c from Thermus thermophilus in Escherichia coli (E. coli) leads to a protein with characteristics of a molten globule. Unfolding induced by guanidine hydrochloride (GdHCl) shows that E. coli-expressed cytochrome c has lower stability (and less cooperativity of unfolding) compared to the protein extracted from Thermus thermophilus, even though the two proteins have identical amino-acid sequences. Moreover, Soret and far-UV circular dichroism signals differ for the two proteins, suggesting a distorted heme environment and more side-chain dynamics of E. coli-expressed cytochrome c. Still, tryptophan fluorescence in E. coli-expressed cytochrome c is quenched as in native protein, and the iron coordinates in a low-spin form. Amino-acid sequencing indicates the presence of only one covalent cysteine-linkage to the heme in E. coli-expressed cytochrome c (normally, there are two linkages), a possible explanation for the trapped, molten-globule-like structure. The features of this non-native protein may be of interest for interpretation of cytochrome c folding kinetics in vitro, since a molten globule may be an intermediate on the folding pathway.     

Successful coping with declining objective life style by institutionalized long-term patients. Results of a follow-up analysis of quality of life in long-term care institutions with the Zurich Quality of Life Inventory

Folding of cytochrome c from its low pH guanidine hydrochloride (Gdn-HCl) denatured state revealed a new intermediate, a five-coordinate high spin species with a water molecule coordinated to the heme. Incorporation of this five-coordinated intermediate into the previously reported ligand exchange model can quantitatively account for the observed folding kinetics. In this new model, unfolded cytochrome c is converted to its native structure through an obligatory folding intermediate, the histidine-water coordination state, whereas the five-coordinate state and a bis-histidine state are off-pathway intermediates. When the concentration of Gdn-HCl in the refolding solution was increased, an acceleration of the conversion from the bis-histidine coordinated state to the histidine-water coordinated state was observed, demonstrating that the reaction requires unfolding of the mis-organized polypeptide structure associated with the bis-histidine state.     

Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism

Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase [CCP(MI)] and horseradish peroxidase (HRP) in their ferric forms. CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl. CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl. The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic. The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl. Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data. The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding. Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture. The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI). Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.     

Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli

We have used thermodynamic and kinetic techniques to monitor the guanidinium chloride induced (GdmCl-induced) denaturation of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli (ePRAI). Although CD-monitored equilibrium denaturation curves are consistent with cooperative unfolding of the protein centered at 1.45 M GdmCl, fluorescence readings drop by over 25% in the region preceding the CD-monitored transition, suggesting non-two-state behavior. Kinetics experiments measure a slow relaxation rate with negative fluorescence amplitude when protein is diluted from 0 to 0.5 M GdmCl, corroborating results from equilibrium conditions. Detection of several unfolding and refolding rates in final GdmCl concentrations from 0 to 5.0 M indicates the presence of at least one intermediate along unfolding and refolding pathways. GdmCl dependence of the relaxation rates can be explained most easily by a nonsequential mechanism for ePRAI unfolding, though a sequential mechanism cannot be ruled out. The data corroborate the fragment complementation studies of Eder and Kirschner [Eder, J., & Kischner, K. (1992) Biochemistry 31, 3617-3625], which are consistent with unfolding of the C-terminal portion of a yeast-derived PRAI in its folding intermediate. In ePRAI, such partial unfolding would expose W391 to quenching by solvent molecules; W356, ePRAI's other tryptophan, is buried in the hydrophobic core and is unlikely to be affected by local changes in structure. A C-terminally unfolded folding intermediate has been demonstrated in the folding of tryptophan synthase (alpha-subunit), a related beta alpha-barrel enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.     

Structural organization in peptide fragments of cytochrome c by heme binding

Prosthetic groups are often important structural organizers of proteins as well as essential functional components. Insertion of prosthetic groups is usually spontaneous, and implies an apoprotein that is partially preorganized to provide a recognition surface for specific binding. Cytochrome c is distinguished by having its heme attached by a dedicated heme lyase through thioether links to cysteine side-chains, and the apoprotein shows no evidence of preorganization under physiological conditions. Nevertheless, addition of heme to two short fragments of cytochrome c enhances helical structure substantially (from approximately 8% to approximately 22%), an effect that depends on iron ligation but not thioether linkage. The helical segments in the corresponding parts of the native holoprotein have little contact surface with heme, implying that the increased helical structure in the fragment complex may depend on tertiary interactions. The absence of the intervening polypeptide chain suggests that the complex represents a relatively independent folded subdomain.     

Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.     

Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.     

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.     

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.     

Characterization of the free energy spectrum of peptostreptococcal protein L

BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration.     

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.     

Steady state and time-resolved fluorescence study of residual structures in an unfolded form of yeast phosphoglycerate kinase

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.     

Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions

The acetic acid-induced unfolding of cytochrome c (cyt c) and apomyoglobin (aMb) are studied under equilibrium conditions by electrospray ionization (ESI) mass spectrometry (MS). The folding states of the proteins in solution are monitored by the charge state distributions that they produce during ESI. A tightly folded protein shows lower charge states than the same protein in an unfolded conformation. The ESI-MS data presented in this study show that during the denaturation of cyt c, only two distinct charge state distributions are observed. These can be attributed to the native and to the acid-unfolded conformation, respectively. In the transition region where the folded and the unfolded conformation are both present in solution, these two distributions are observed simultaneously, thus giving rise to a bimodal ESI mass spectrum. These data reflect a highly cooperative (two state) folding behavior. In contrast, the acid-induced unfolding of aMb is accompanied by gradual shifts in the maxima of the observed charge state distribution. This indicates a non-cooperative unfolding behavior involving multiple protein conformations. The observations made here suggest that ESI-MS might be a general method for assessing the cooperativity of protein unfolding transitions. This study also addresses the issue of 'secondary' solvent effects for ESI-MS studies on the acid-induced unfolding of proteins. These effects influence the ESI charge state distribution without being related to conformational changes of the protein in solution and could potentially complicate the interpretation of ESI mass spectra. Data obtained for bovine pancreatic trypsin inhibitor and ubiquitin indicate that secondary solvent effects influence the observed charge state distributions only to a very minor extent between pH 8.5 and 2.5.     

Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride

The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).     

Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.     

Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding

The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.     

Refolding of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase in the presence of ligand: a stopped-flow NMR spectroscopy study

Escherichia coli dihydrofolate reductase contains five tryptophan residues that are spatially distributed throughout the protein and located in different secondary structural elements. When these tryptophans are replaced with [6-19F]tryptophan, distinct native and unfolded resonances can be resolved in the 1-D 19F NMR spectra. Using site-directed mutagenesis, these resonances have been assigned to individual tryptophans [Hoeltzli, S. D., and Frieden, C. (1994) Biochemistry 33, 5502-5509], allowing both the native and unfolded environments of each tryptophan to be monitored during the refolding process. We have previously used these assignments and stopped-flow NMR to investigate the behavior of specific regions of the protein during refolding of apo dihydrofolate reductase from urea in real time. These studies now have been extended to investigate the real time behavior of specific regions of the protein during refolding of dihydrofolate reductase in the presence of either NADP+ or dihydrofolate. As observed for the apoprotein, in the presence of either ligand, unfolded resonance intensities present at the first observed time point (1.5 s) disappear in two phases similar to those monitored by either stopped-flow fluorescence or circular dichroism spectroscopy. The existence of unfolded resonances which disappear slowly indicates that an equilibrium exists between the unfolded side chain environment and one or more intermediates, and that formation of at least one intermediate is cooperative. The results of this study are consistent with previous fluorescence studies demonstrating that dihydrofolate binds at an earlier step in the folding process than does NADP+ [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413-4416] and provide a structural interpretation of the previous results. In the apoprotein as well as in the presence of either ligand, the protein folds via at least one cooperatively formed, solvent-protected intermediate which contains secondary structure. In the presence of NADP+, a stable native-like side chain environment forms in the regions around tryptophans 30, 133, and 47 in an intermediate which cannot bind NADP+ tightly. Native side chain environment forms in the regions around tryptophans 22 and 74 only in the structure which is able to bind NADP+ tightly. In the presence of dihydrofolate, stable native-like side chain environment forms cooperatively in the regions around each tryptophan in a non-native intermediate which must undergo a conformational change prior to binding NADP+. The presence of ligands influences the processes which occur during the folding of dihydrofolate reductase, and the ligand may in effect serve as part of the hydrophobic core.     

Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.