pH-jump-induced folding and unfolding studies of barstar: evidence for multiple folding and unfolding pathways.

Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.     

pH dependent conformational and structural changes of xylanase from an alkalophilic thermophilic Bacillus sp (NCIM 59)

The pH induced conformational and structural changes of Xyl II have been investigated from the alkalophilic thermophilic Bacillus sp. using kinetic, circular dichroism and fluorescence spectroscopy studies. The systematic studies on the folding and stability of cellulase-free xylanases are important, since their biotechnological applications require them to function under extremes of pH and temperature. The Trp fluorescence and the kinetic constants were found dependent on the pH. Above pH 8, the enzyme exhibited unfolding transitions as revealed by a red shift in the emission maximum as well as decreases in the fluorescence intensity. Circular dichroism studies revealed a decrease in the CD ellipticity at 222 nm at pH 9 and 10. The reduced catalytic activity of Xyl II at alkaline pH is correlated to the pH induced unfolding and ionization or protonation of key protein residues. The pH profile of Xyl II showed apparent pK values of 5.5 and 7 for the free enzyme and 5.6 and 6.7 for the enzyme-substrate complex. The abnormally high pK of 6.7 indicated the participation of a carboxyl group present in a non-polar environment. The pH dependence of inactivation kinetics of Xyl II with Woodward's reagent K corroborates evidence for the presence of a catalytically important carboxyl residue. The sequence alignment studies of Xyl II, in combination with kinetic and chemical modification data provide strong evidence for the participation of Asp94 in the catalytic function. The Xyl II produced from an alkalophilic source, was stable at pH 10 with a t(1/2) of 24 h. However, the enzyme exhibited pH optimum at near neutral values, which can be explained by the ionization and microenvironment of the active site residues.     

Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase

To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.     

The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.     

Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25 degrees C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.     

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi‐domain trimeric α‐helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two‐state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter‐subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate‐limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB_Δloop, from the SDS unfolded state. The CD spectrum of the refolded AcrB_Δloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re‐association of the trimer might be the limiting factor to obtain folded wild‐type AcrB.     

Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding

The CL fragment of a type-kappa immunoglobulin light chain in which the C-terminal cysteine residue was modified with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (CL-AEDANS fragment) was prepared. This fragment has only one tryptophan residue at position 148. The compactness of the fragment whose intrachain disulfide bond was reduced in order for the tryptophan residue to fluoresce (reduced CL-AEDANS fragment) was studied in the early stages of refolding from 4 M guanidine hydrochloride by fluorescence energy transfer from Trp 148 to the AEDANS group. The AEDANS group attached to the SH group of a cysteine scarcely fluoresced when excited at 295 nm. For the reduced CL-AEDANS fragment, the fluorescence emission band of the Trp residue overlapped with the absorption band of the AEDANS group, and the fluorescence energy transfer was observed between Trp 148 and the AEDANS group in the absence of guanidine hydrochloride. In 4 M guanidine hydrochloride, the distance between the donor and the acceptor was larger, and the efficiency of the energy transfer became lower. The distance between Trp 148 and the AEDANS group for the intact protein estimated by using the energy-transfer data was in good agreement with that obtained by X-ray crystallographic analysis. By the use of fluorescence energy transfer, tryptophyl fluorescence, and circular dichroism at 218 nm, the kinetics of unfolding and refolding of the reduced fragment were studied. These three methods gave the same unfolding kinetic pattern. However, the refolding kinetics measured by fluorescence energy transfer were different from those measured by tryptophyl fluorescence and circular dichroism, the latter two giving the same kinetic pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.     

Structural characterization and unfolding mechanism of human 4F2hc ectodomain

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)(8) barrel; C, antiparallel β(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.     

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.     

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.     

Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.     

Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis

β₂-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of β2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of β2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V β2-ms) as well as the wild-type β2-m. W39 β2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 β2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V β2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.