Mechanism of acid-induced folding of proteins

We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulation of the acidic compact state of apomyoglobin from yellowfin tuna

A molecular model of the acidic compact state of apomyoglobin (A-state) from yellowfin tuna was obtained using molecular dynamics simulations (MD) by calculating multiple trajectories. To cause partial unfolding within a reasonable amount of CPU time, both an acidic environment (pH 3 and 0.15M NaCl) and a temperature jump to 500 K were needed. Twenty-five acidic structures of apomyoglobin were generated by MD, 10 of them can be clustered by RMSD in an average structure having a common hydrophobic core as was reported for acidic sperm whale apomyoglobin, with shortened helices A,G,E, and H (the helix A appears to be translated along the sequence). Prolonging the MD runs at 500 K did not cause further substantial unfolding, suggesting that the ensemble of generated structures is indicative of a region of the conformational space accessible to the apoprotein at acidic pH corresponding to a local energy minimum. The comparison of experimentally determined values of specific spectroscopic properties of the apomyoglobin in acidic salt conditions with the expected ones on the basis of the MD generated structures shows a reasonable agreement considering the characteristic uncertainties of both experimental and simulation techniques. We used frequency domain fluorometry, acrylamide fluorescence quenching, and fluorescence correlation spectroscopy together with far UV circular dichroism to estimate the helical content, the Stern-Volmer quenching constant and the radius of gyration of the protein. Tuna apomyoglobin is a single tryptophan protein and thus, interpretation of its intrinsic fluorescence is simpler than for other proteins. The high sensitivity of the applied fluorescence techniques enabled experiments to be performed under very dilute conditions, that is, at concentrations of subnanomolar for the FCS measurements and 6 muM for the other fluorescence measurements. As high concentrations of proteins can strongly affect the association equilibrium among partially unfolded states, fluorescence techniques can provide complementary information with respect to other techniques requiring higher sample concentrations, such as NMR. The analysis of exposed hydrophobic regions in each of the MD-generated acidic structures reveals potential candidates involved in the aggregation processes of apomyoglobin in the acidic compact state. Our investigation represents an effective model system for studying amyloid fibril formation found in important diseases that are believed to proceed via aggregation of protein in the molten globule state.     

The effect of molecular confinement on the conformational dynamics of the native and partly folded state of apomyoglobin.

Inclusion in agarose gel significantly affects the conformational dynamics of native and acidic partly folded states of tuna apomyoglobin, a single tryptophan containing protein, as documented by frequency domain fluorometry investigations. The heterogeneity of the tryptophanyl emission decay increases on gel inclusion compared to that observed for free-in-solvent protein at both neutral and acidic pH, thus suggesting that the interconversion rate among conformational substates is somewhat reduced. The observation that this effect is much more pronounced for the partly folded state suggests that confined environments such as those existing in the living cells might favor the sequential folding process avoiding that structured intermediates rapidly convert into less structured ones.     

Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis

The individual emission properties of the two tryptophanyl residues of sperm-whale apomyoglobin have been resolved by examining the fluorescence variations induced by denaturants, i.e., acid and guanidine, on apomyoglobin mutants W7F and W14F. The fluorescence changes have been correlated to the conformational transitions undergone by apomyoglobin on increasing denaturant concentration. The results indicate that the fluorescence decrease, observed for sperm-whale apomyoglobin on going from pH 8.0 to pH 6.0, cannot be ascribed to the formation of a charge transfer complex between a nearby histidine residue and W14 as reported in earlier papers but rather to minor structural changes affecting the microenvironments of both residues. The formation of the acidic partly folded state around pH 4.0 determines an increase of the fluorescence yield and a small red shift (5 nm) of W7 due to removal of sterically interacting K79, which is able to attenuate the emission of this residue in the native state. The fluorescence intensity of the other residue, i.e., W14, is not affected by the acidic transition. Guanidine denaturation experiments revealed an increase of fluorescence yield of W14 upon the intermediate formation, whereas the fluorescence of the other residue remained constant. The results suggest that the unfolding pathway may be different depending on the chemical nature of the denaturant used.     

Mechanism of the conformational transition of melittin.

It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational transition of melittin, we examined in detail the conformation of melittin under various conditions by far-UV circular dichroism at 20 degrees C. We found that the helical conformation is also stabilized by strong acids such as perchloric acid. The effects of various acids varied largely and were similar to those of the corresponding salts, indicating that the anions are responsible for the salt- or acid-induced transitions. The order of effectiveness of various monovalent anions was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding is responsible for the salt- or acid-induced transitions. From the NaCl-, HCl-, and alkaline pH-induced conformational transitions, we constructed a phase diagram of the anion- and pH-dependent conformational transition. The phase diagram was similar in shape to that of acid-denatured apomyoglobin [Goto, Y., & Fink, A.L. (1990) J. Mol. Biol. 214, 803-805] or that of the amphiphilic Lys, Leu model polypeptide [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396], suggesting a common mechanism of the conformational transition. The anion-, pH-, and peptide concentration-dependent conformational transition of melittin was explained on the basis of an equation in which the conformational transition is linked to proton and anion binding to the titratable groups.     

Thermodynamic study of the apomyoglobin structure

Sperm whale apomyoglobin has been studied thermodynamically in solutions with different pH and temperature by scanning microcalorimetry, viscosimetry, nuclear magnetic resonance and circular dichroism spectrometry, and by electrometric and calorimetric titration. It has been shown that apomyoglobin in solutions with pH close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is maximally stable at about 30 degrees C and breaks down reversibly both upon heating or cooling from this temperature. The process of breakdown of this structure is highly co-operative and can be regarded as a transition between two macroscopic states of protein, the native and denatured states. In contrast to the native state, which is specified by definite values of compactness and ellipticity, the compactness and ellipticity of the denatured state of apomyoglobin depend strongly on pH; with a decrease of pH below 4.0, these parameters gradually approach the values of the random coil.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Molecular confinement influences protein structure and enhances thermal protein stability

The sol-gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, alpha-lactalbumin, and metmyoglobin retained native-like solution structures following sol-gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the alpha-helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.     

Structural details,pathways, and energetics of unfolding apomyoglobin

Protein folding is often difficult to characterize experimentally because of the transience of intermediate states, and the complexity of the protein-solvent system. Atomistic simulations, which could provide more detailed information, have had to employ highly simplified models or high temperatures, to cope with the long time scales of unfolding; direct simulation of folding is even more problematic. We report a fully atomistic simulation of the acid-induced unfolding of apomyoglobin in which the protonation of acidic side-chains to simulate low pH is sufficient to induce unfolding at room temperature with no added biasing forces or other unusual conditions; and the trajectory is validated by comparison to experimental characterization of intermediate states. Novel insights provided by their analysis include: characterization of a dry swollen globule state forming a barrier to initial unfolding or final folding; observation of cooperativity in secondary and tertiary structure formation and its explanation in terms of dielectric environments; and structural details of the intermediate and the completely unfolded states. These insights involve time scales and levels of structural detail that are presently beyond the range of experiment, but come within reach through the simulation methods described here. An implicit solvation model is used to analyze the energetics of protein folding at various pH and ionic strength values, and a reasonable estimate of folding free energy is obtained. Electrostatic interactions are found to disfavor folding.     

EFFECT OF SALTS ON THE PHYSICAL AND KINETIC PROPERTIES OF HUMAN PLACENTAL CHOLINE ACETYLTRANSFERASE

Abstract— The effects of salt on the properties of human placental choline acetyltransferase have been examined. Increases in enzyme activity, thermal denaturation and susceptibility to proteolysis can be related to increases in ionic strength, rather than to specific salt effects. Increased ionic strength increases the maximal velocity (K_m) of the reaction, with no change in the kinetic parameter V_max/K_m (choline). The pH-K_m profile, measured over the range of 6.5–8.0, indicates the requirement of a dissociated acidic residue whose pKa is below 7.5 at high ionic strength, and a protonated residue whose pKa is above 7.5 at low ionic strength. It is proposed that the conformation of the enzyme is different at high ionic strength and at low ionic strength, and that these different conformational states of the enzyme result in different rate-determining steps of the reaction.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Interactions of apomyoglobin with membranes: mechanisms and effects on heme uptake

The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged phospholipids. The pH dependence of apomyoglobin interactions with membranes is a two-step process, and involves a partially folded state stabilized at acidic pH. An evident role for the interaction of apomyoglobin with lipid bilayers would be to facilitate the uptake of heme from the outer mitochondrial membrane. However, heme binding to apomyoglobin is observed at neutral pH when the protein remains in solution, and slows down as the pH becomes more favorable to membrane interactions. The effective incorporation of soluble heme into apomyoglobin at neutral pH suggests that the interaction of apomyoglobin with membranes is not necessary for the heme uptake from the lipid bilayer. In vivo, however, the ability of apomyoglobin to interact with membrane may facilitate its localization in the vicinity of the mitochondrial membranes, and so may increase the yield of heme uptake. Moreover, the behavior of apomyoglobin in the presence of membranes shows striking similarities with that of other proteins with a globin fold. This suggests that the globin fold is well adapted for soluble proteins whose functions require interactions with membranes.     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

Thermodynamic study of the structure of apomyoglobin

Sperm whale apomyoglobin structure has been studied thermodynamically at different temperatures and pH of solution by scanning microcalorimetry, viscosimetry, NMR and CD spectrometry techniques. It has been shown that at pH close to neutral, apomyoglobin has a compact highly cooperative structure with a well defined hydrophobic core. The stability of this structure is maximal at 30 degrees C and decreases both with an increase and decrease of temperature. Correspondingly, the compact three-dimensional structure of apomyoglobin is disrupted both upon heating and cooling of the solution. In acidic solutions this process is reversible and represents a cooperative transition between two macroscopic states--the ordered and disordered ones which can be regarded as the native and denatured states of molecule. The compactness and ellipticity of the denatured state depend significantly on pH: upon a decrease of pH in the region of ionization of carboxylic groups these parameters approach the values characteristic of a random coil. A comparison of the maximal stability of the cooperative structure of apomyoglobin which is 12 kJ.mol-1 at 30 degrees C and pH close to neutral ones with the maximal stability of metmyoglobin which is 49 kJ.mol-1 shows that the contribution of heme in the stabilization of the native myoglobin structure reaches 37 kJ.mol-1.     

Pressure-induced perturbation of ANS-apomyoglobin complex: frequency domain fluorescence studies on native and acidic compact states.

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.     

The conformational manifold of ferricytochrome c explored by visible and far-UV electronic circular dichroism spectroscopy

The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and non-native states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed III h, which is structurally different from the alkaline states, IV 1 and IV 2, contrary to current belief. At neutral or slightly acidic pH, III h is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm (-1), which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, III h, does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe (3+)-M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice.     

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.     

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.     

The role of the salt concentration, proton, and phosphate binding on the thermal stability of wild and cloned DNA-binding protein Sso7d from Sulfolobus solfataricus

The acidic pH (1.5-7.0) and ionic strength (0.005-0.2M) dependence of thermodynamic functions of protein Sso7d from Sulfolobus solfataricus, cloned (c-Sso7d) and N-heptapeptide deleted [c-des(1-7)Sso7d] in glycine, and phosphate buffers was studied by means of adiabatic scanning calorimetry. The difference of proton binding was estimated from deltaHcal(pH), Td(pH), and (deltaTd/deltapH). It was found that a single group non-co-operative ionization with apparent pKa = 3.25 for both cloned and deleted proteins govern the thermal unfolding of two different (protonated and unprotonated) forms. deltaH degrees is found to be pH-independent and the changes in stability (deltaG degrees ) originate from changes in entropy terms. The apparent pKa measured at high salt concentrations decreases with 0.5 pH units from glycine to phosphate and the free energy of transfer at high ionic strength is 0.7 kcal/mol. The ionic strength dependence for the pH-dependent D-states is very different at pH 6.0 and 1.5. This is consistent with the property of denatured state to be more compacted or "closed" (Dc) at neutral or weak acidic pH and more random or "open" (Do) at acidic pH. From the Bjerrum's relation was found the number of screened charges important for the unfolding process. The main conclusions are: (1) the thermal stability of Sso7d has prominently entropic nature; (2) a single non-co-operative ionization controls the conformations in the D-state; and (3) pH-dependent conformational equilibrium could be functionally important in Sso7d-DNA recognition.