Acid-denatured State Research Articles

Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor.

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.

Effect of H helix destabilizing mutations on the kinetic and equilibrium folding of apomyoglobin

Acid-denatured apomyoglobin (apoMb) contains residual helical structure in the region of the polypeptide which corresponds to the H helix of the folded protein. In order to elucidate the role of this residual secondary structure in the protein folding process and to determine whether residual structure in the denatured state affects either the overall rate of folding or the rate of formation of a burst phase intermediate, we have examined the equilibrium and kinetic folding behavior of a mutant designed to destabilize residual secondary structure in the H helix region. Both Asn132 and Glu136 were changed to Gly (N132G,E136G) to effect this destabilization. Circular dichroism spectra show that the mutant protein contains less helical structure in the acid-denatured state and in the equilibrium intermediate state at pH 4.2 than does the wild-type protein. The CD spectra of the native states of the two proteins are nearly identical. The refolding kinetics for each of the species were measured by stopped-flow CD in the far-UV region and by NMR quench-flow pulse labeling. Under identical conditions, the CD-detected refolding of wild-type and mutant apomyoglobin from the acid-denatured state or from the urea-denatured state occurs at very similar rates following a burst phase that occurs too rapidly to measure by the stopped-flow technique. The urea dependence of the unfolding and refolding rates is consistent with the presence of at least one obligatory on-pathway intermediate in both wild-type and mutant proteins. The kinetic intermediate of the mutant protein is considerably less stable than that of the wild-type protein. Hydrogen exchange pulse labeling experiments indicate that, in contrast to the wild-type protein, the H helix is not stabilized during the burst phase refolding of the mutant but becomes stabilized during the slower phases. While the wild-type and mutant proteins both form compact intermediates, these differ in the content and location of secondary structure. The rate of folding of the AGH subdomain, which takes place prior to the transition state, is substantially slower for the N132G,E136G mutant protein. A strong propensity for spontaneous formation of helical structure in the H helix region is not a prerequisite for efficient folding nor for formation of equilibrium or kinetic intermediates. These observations suggest that while folding of apomyoglobin proceeds through an obligatory intermediate, the precise structure of this intermediate is not critical and its secondary structure may be altered without substantially affecting either the overall refolding kinetics or the integrity of the final folded state.

Conformational Properties of the A-State of Cytochrome c Studied by Hydrogen/Deuterium Exchange and Electrospray Mass Spectrometry

Hydrogen/deuterium (H/D) exchange studies that were monitored by liquid chromatography–electrospray ionization mass spectrometry (LC–ESIMS) were used to obtain a structural description of the compact acid-denatured state of ferricytochrome c (A-state). Due to the very different solvent conditions necessary to generate the nonnative states, it was essential that after deuterium labeling the nonnative states were refolded to the native state to insure high reproducibility during sample preparation and LC–ESIMS analysis. Approximately 30% lower deuterium was found incorporated in the A-state compared to the acid-denatured (UA) state. The analysis of the width of the mass peak suggests that the distribution of conformers sampled in the A-state was relatively narrow and that the compactness of the A-state was much closer to that of the native state than to the acid-denatured state. The LC–ESIMS study of partially deuterium-labeled peptic fragments derived from the A-state conformer generated under H/D quenching conditions were interpreted in terms of a significant loss of structural integrity within amino acid region 22–46.

The Methanol-induced Globular and Expanded Denatured States of Cytochrome c: A Study by CD Fluorescence, NMR and Small-angle X-ray Scattering

Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering. At pH 3.0 and 25°C, two methanol-induced non-native states were characterized. First, addition of methanol up to 25% (v/v) induced a compact denatured conformer (I M). Further addition of methanol transformed this I Mstate into the expanded and highly helical denatured state (H). The existence of the I Mstate was shown by the discrepancy in transition curves obtained from the ellipticity at 222 nm, the ellipticity at 282 nm, the tryptophan fluorescence monitored at 350 nm and the native peak intensity of the 1H NMR spectrum. These CD, fluorescence and NMR results showed that the I Mstate has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state. The radius of gyration of the I Mstate, 17.7 Å, obtained from the Guinier plot of the small-angle X-ray scattering data was significantly smaller than that of the acid-denatured state (30.1 Å) and was closer to that of the native state (14.6 Å), showing that the I Mstate is compact. The Kratky plot for the I Mstate exhibited a bell-shaped profile, indicating a globular conformation. These structural features indicate that the structure of the I Mstate is quite similar to that of the anion-induced molten globule state of this protein. Furthermore the alcohol-denatured state (H) of cyt cin 60% (v/v) methanol was structurally characterized. Though the H state had a helical content much higher than the native state monitored by far-UV CD spectroscopy, the radius of gyration, 31.7 Å, was similar to that of the acid-denatured state, showing that this H state is an expanded denatured state. The Kratky plot for the H state did not show a clear peak, indicating a chain-like conformation. Thus we conclude that the H state has an expanded and chain-like conformation with a high helical content. Finally, we constructed a phase diagram of cyt cinvolving the native, I M, acid-denatured and H states against pH and the methanol concentration. The result indicates that the I Mstate is found in the pH range from 2.5 to at least 4.5 with a pH-dependent optimum methanol concentration of 10 to 40%.

Spectroscopic and volumetric investigation of cytochrome c unfolding at alkaline pH: characterization of the base-induced unfolded state at 25 degrees C.

We have measured at 25 degrees C the relative specific sound velocity increment, [u], and the partial specific volume, v degrees, of cytochrome c as a function of pH. Our data reveal that the base-induced native to unfolded transition of the protein is accompanied by a volume decrease of 0.014 cm3 g-1 and a compressibility decrease of 3.8 x 10(-6) cm3 g-1 bar-1. These results allow us to conclude that, relative to a fully unfolded conformation, the base-denatured state of cytochrome c has only 70 to 80% of its surface area exposed to the solvent. Recently, we reported a similar result for the acid-denatured state of cytochrome c. Thus, insofar as solvent exposure is concerned, both the base- and the acid-induced unfolded states of cytochrome c retain some order, with 20 to 30% of their surface areas remaining solvent-inaccessible. We discuss the implications of this result in terms of defining potential intermediate states in protein folding pathways.

The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy.

In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in delta 131 delta, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meander may be highly populated in delta 131 delta, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand beta 1 in delta 131 delta also disappear, suggesting that beta 1 may combine with the beta 2-beta 3 hairpin to form a native-like beta meander. In addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to delta 131 delta at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.

Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase.

Prolyl oligopeptidase is the prototype of a new serine protease family, unrelated to trypsin and subtilisin. In contrast with these proteases, prolyl oligopeptidase is remarkably sensitive to ionic strength, being more active in the presence of high concentrations of salt. The enzyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M NaCl on the stability of the enzyme were investigated by studying its denaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that denaturation of the enzyme is promoted in the presence of NaCl. Furthermore, destabilization of the low-pH form by urea is more significant than that of the high-pH form. Examination of the fluorescence emission spectra of various denatured forms indicates that the enzyme is not fully unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interpreted in terms of the decay of the protective water shell at the higher ionic strength. The higher enthalpy and entropy of activation for heat denaturation provide further evidence that a more ordered water structure stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme has two domains of different stabilities.

A Comparison of the pH, Urea, and Temperature-denatured States of Barnase by Heteronuclear NMR: Implications for the Initiation of Protein Folding

The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile→Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3 JNH-C αH coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of β-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the β-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites may be, and the kinetic and protein engineering studies show which ones are productive.

Equilibrium folding studies of cellular retinoic acid binding protein, a predominantly beta-sheet protein.

We have examined the conformational behavior under various unfolding conditions of a predominantly beta-sheet protein, cellular retinoic acid binding protein (CRABP). Urea unfolding-refolding of CRABP is a highly cooperative process that can be approximated by a two-state model. Acid denaturation is also cooperative and reversible and leads to a state containing nonnative residual structure: Below pH 2.6, CRABP contains a substantially larger amount of alpha-helix than under native conditions. CRABP adopts up to 75% alpha-helix in solutions containing a high percentage of 2,2,2-trifluoroethanol. The acid-denatured state of CRABP undergoes a conformational change to a state containing predominantly beta-sheet structure upon the addition of small amounts of Na2SO4. This conformational malleability may be important for the folding mechanism of CRABP. The possible implication of nonnative alpha-helical structure in the folding of CRABP is discussed.

Determining stability of proteins from guanidinium chloride transition curves.

The guanidinium chloride (GdmCl) denaturation of RNAase A, lysozyme and metmyoglobin was investigated at several pH values by using absorbance measurements at 287, 300 and 409 nm respectively. From these measurements the free-energy change on denaturation, delta Gapp., was calculated, assuming a two-state mechanism, and values of delta Gapp. at zero concentration of the denaturant were measured. For each protein all delta Gapp. values were adjusted to pH 7.00 by using the appropriate relationship between delta Gapp. and pH. Dependence of the adjusted delta Gapp. value on GdmCl concentration increases for metmyoglobin and decreases for the other two proteins as the denaturant concentration decreases. It has been shown that these are expected results if the presence of the acid-denatured state during the GdmCl denaturation of proteins is considered.

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.

Effect of Amino-Terminal Processing byStaphylococcus aureusV-8 Protease on Activity and Structure of Recombinant Human Interferon-γ

Treatment of recombinant human interferon-gamma (rHuIFN-gamma) with Staphylococcus aureus V-8 protease generated a transiently stable species that lacks 10 amino-terminal residues. This protein showed distinct secondary and higher-order structures with an alpha-helical content of 31%, suggesting that the secondary and tertiary structure largely remain upon removal of 10 amino-terminal residues. The antiviral activity was abolished, or greatly reduced, for this species relative to the intact protein. These results suggest an important role for the amino-terminal portion in the activity of human interferon-gamma. Since the digested protein is difficult to refold from the acid-denatured state, it was concluded that, although not essential for maintaining the core structure of the protein, the amino-terminal portion is critical for refolding the protein from acid.

Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase .beta.2 subunit

A reversible acid-denaturation process of the beta 2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of beta 2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native beta 2. It is shown that the association occurs very early in the folding of beta 2. The association rate constants of the antibody with the immunoreactive folding intermediate and with native beta 2 are the same (3 X 10(5) M-1.s-1). But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 X 10(-2) s-1) isomerization of refolding beta chains. This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of beta 2. Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope.

Effects of sodium dodecyl sulfate on the structure of histones H1

The effects of sodium dodecyl sulfate (SDS) on the structure of histones H1 as model proteins have been studied by a combination of difference spectroscopy, circular dichroism (CD), and spectrofluorometry. The detergent increases the α-helix content at the expense of random-coiled regions. As measured by CD, this transition involves 44–50 residues in calf H1. Assuming that positive charges in the amino acid side chains are no longer an impediment to the formation of α-helix in the presence of SDS, the use of the method of prediction of secondary structure elaborated by Chou and Fasman gives an estimate of six regions with high helix-forming potential. One of these peptides lies in the NH2-terminal region (residues 22–29), whereas the five remaining peptides are in the COOH-terminal region of the histone (residues 109–114, 120–125, 142–151, 185–189, and 202–211). These six peptides amount to 45 residues, in good agreement with experimental results. We have also studied the action of the detergent on the environment of tyrosyl residues of calf H1 (one tyrosine) andCeratitis capitata H1 (two tyrosines). Difference spectroscopy and CD show that the environment of tyrosine-72 of calf H1 in the histone-SDS complex differs from both the native state and the acid-denatured state. The two tyrosyl residues ofCeratitis H1, whose environments in the native protein are markedly different, are included in similar environments in the histone-SDS complex.