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C A Royer

Publications and source records attributed to C A Royer.

At least 19 recordsLinked to original sources
PubMed1999

Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy.

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, l-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR. For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression.

Amino Acid Substitution↗
PubMed1999

Pressure-jump studies of the folding/unfolding of trp repressor.

The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.

Bacterial Proteins↗
PubMed1999

Probing the physical basis for trp repressor-operator recognition.

The bacterial repressor protein, trp repressor, is one of the best studied transcriptional regulatory proteins in terms of function, structure, dynamics and stability. Despite these significant advances, the structural and energetic basis for the specific recognition of its operator sites by trp repressor remains poorly understood. In fact, recognition in this system is controled by the binding of the co-repressor ligand, l-tryptophan, as well as by conformational and dynamic properties of the operator targets, DNA sequence-dependent control of the oligomerization properties of the repressor, water-mediated interactions, and specific interactions involving the peptide backbone and phosphate moieties. Moreover, only one direct contact between the protein and the DNA is evident from the crystallographically determined structure of the complex. In an attempt to better define how the various sequence elements in the operator target contribute to this complex control of affinity and cooperativity of trp repressor binding, we have studied the binding of trp repressor to a series of mutated operator targets using fluorescence anisotropy, which provides very high quality data allowing fairly precise estimations of the affinities involved. We conclude from these studies that even on very small (25 bp) targets, the repressor binds slightly cooperatively, populating a 2:1 dimer/DNA complex, and then at higher concentrations a third dimer is bound with significantly lower affinity, revealing an inherent asymmetry in the trpEDCBA-derived target. Investigation of the basis for the asymmetry implicates the identity of the second base in the so-called structural half-site GNACT, which apparently influences the switch between tandem and simple binding. Mutation of the C or the T bases in the structural half-site abolishes all specificity in binding, and alteration of the single direct contact, the G of the structural half-site, or the central TTAA significantly lowers the affinity of the dimer for its site, without modifying the apparent cooperativity. Finally, we note that the order of affinity is conserved in the absence of the co-repressor, and moreover, it is in all cases significantly higher than that observed for holo-repressor binding to non-specific DNA, indicating that one cannot simply equate apo-repressor and non-specific binding.

Bacterial Proteins↗
PubMed1999

Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor.

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-423) was expressed as an amino-terminal His-tagged protein in a bacterial expression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its ligand, 1,25-dihydroxyvitamin D3, with high affinity, similar to that of the full-length protein. Saturation of the protein with ligand quenched 90% of the tryptophan fluorescence, consistent with the purified protein being uniformly able to bind ligand. Addition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal following the addition of ligand, consistent with a change in the environment of aromatic amino acid side chains. The far-UV circular dichroism spectrum was consistent with a protein of high alpha-helical content. Sedimentation equilibrium experiments demonstrated that the protein formed higher-order complexes, and the distribution of the protein among these complexes was significantly shifted by addition of ligand.

Animals↗
PubMed1999

Exploring the temperature-pressure phase diagram of staphylococcal nuclease.

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I' and tyrosine O-H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase.

Kinetics↗
PubMed1999

Probing the role of water in the tryptophan repressor-operator complex.

The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions.

Bacterial Proteins↗
PubMed1998

Structural characterization of the pressure-denatured state and unfolding/refolding kinetics of staphylococcal nuclease by synchrotron small-angle X-ray scattering and Fourier-transform infrared spectroscopy.

The pressure-induced unfolding of wild-type staphylococcal nuclease (Snase WT) was studied using synchrotron X-ray small-angle scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy, which monitor changes in the tertiary and secondary structural properties of the protein upon pressurization. The experimental results reveal that application of high-pressure up to 3 kbar leads to an approximate twofold increase of the radius of gyration Rg of the native protein (Rg approximately 17 A) and a large broadening of the pair-distance-distribution function, indicating a transition from a globular to an ellipsoidal or extended chain structure. Analysis of the FT-IR amide I' spectral components reveals that the pressure-induced denaturation process sets in at 1.5 kbar at 25 degrees C and is accompanied by an increase in disordered and turn structures while the content of beta-sheets and alpha-helices drastically decreases. The pressure-induced denatured state above 3 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as a fully extended random coil. Temperature-induced denaturation involves a further unfolding of the protein molecule which is indicated by a larger Rg value and significantly lower fractional intensities of IR-bands associated with secondary-structure elements. In addition, we have carried out pressure-jump kinetics studies of the secondary-structural evolution and the degree of compactness in the folding/unfolding reactions of Snase. The effect of pressure on the kinetics arises from a larger positive activation volume for folding than for unfolding, and leads to a significant slowing down of the folding rate with increasing pressure. Moreover, the system becomes two-state under pressure. These properties make it ideal for probing multiple order parameters in order to compare the kinetics of changes in secondary structure by pressure-jump FT-IR and chain collapse by pressure-jump SAXS. After a pressure jump from 1 bar to 2.4 kbar at 20 degrees C, the radius of gyration increases in a first-order manner from 17 A to 22.4 A over a timescale of approximately 30 minutes. The increase in Rg value is caused by the formation of an extended (ellipsoidal) structure as indicated by the corresponding pair-distance-distribution function. Pressure-jump FT-IR studies reveal that the reversible first order changes in beta-sheet, alpha-helical and random structure occur on the same slow timescale as that observed for the scattering curves and for fluorescence. These studies indicate that the changes in secondary structure and chain compactness in the folding/unfolding reactions of Snase are probably dependent upon the same rate-limiting step as changes in tertiary structure.

Kinetics↗
PubMed1998

Probing the contribution of internal cavities to the volume change of protein unfolding under pressure.

The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding.

Micrococcal Nuclease↗
PubMed1998

Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states.

The volume change for the transition from the native state of horse heart apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protein by pressure at pH 4.2 revealed an upper limit for the unfolding of the intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the entire transition from native to molten globule to unfolded state was observed in the available pressure range below 2.5 kbar. The volume change for the N-->I transition is relatively large and does not correlate well with the changes in relative hydration for these transitions derived from measurements of the changes in heat capacity, consistent with the previously observed lack of correlation between the m-value for denaturant-induced transitions and the measured volume change of unfolding for cooperativity mutants of staphylococcal nuclease (Frye et al. 1996. Biochemistry. 35:10234-10239). Our results support the hypothesis that the volume change associated with the hydration of protein surface upon unfolding may involve both positive and negative underlying contributions that effectively cancel, and that the measured volume changes for protein structural transitions arise from another source, perhaps the elimination of void volume due to packing defects in the structured chains.

Animals↗
PubMed1998

Visualization of trp repressor and its complexes with DNA by atomic force microscopy.

We used tapping mode atomic force microscopy to visualize the protein/protein and the protein/DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. In the presence of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mica surface. We observed the expected decrease in specificity of TR for its operators with increasing protein concentration (1-5 nM). This loss of DNA-binding specificity is accompanied by the formation of large protein assemblies of varying sizes on the mica surface, consistent with the known tendency of the repressor to oligomerize in solution. When the co-repressor is omitted, no repressor molecules are seen, either on the plasmid fragments or free on the mica surface, probably because of the formation of larger aggregates that are removed from the surface upon washing. All these findings support a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor.

Bacterial Proteins↗
PubMed1997

Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy.

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Animals↗
PubMed1997

Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides.

Fluorescence-based solution methods have been used to study the binding of the trp repressor of Escherichia coli to a series of oligonucleotides bearing all or partial determinants for high affinity specific binding. The tryptophan, salt concentration and competitor DNA dependence of the binding affinities was examined for these targets. Binding to a fluorescein-labeled 20 base-pair hairpin structure oligonucleotide, which contains a palindromic repressor binding site (GAACTAGTTAACTAGTAC) and is known to bind repressor in a 1 : 1 dimer-DNA complex, resulted in a protein concentration-dependent, competable static quenching of fluorescence in presence of co-repressor, l-tryptophan. The affinity recovered from the fits of these intensity profiles at 100 mM KCl was on the order of 4x10(8) M-1. In absence of co-repressor an increase in intensity at high repressor concentration (>10(-7) M) was observed. The salt concentration dependence of the specific binding of the holo-repressor to this oligonucleotide was approximately half as large as what would be predicted by the number of phosphate contacts in the crystal structures of the complex. Repressor binding to the fluorescein-labeled hairpin 20mer was compared with binding to a rhodamine-labeled 36 base-pair oligonucleotide bearing two inverted structural half-sites GNACT separated by an eight base-pair spacer containing none of the natural intervening sequence. The rather low affinity observed for the 36mer revealed that the intervening sequence in the natural operators contains energetic specificity determinants. Binding to a rhodamine-labeled oligonucleotide bearing a completely non-specific sequence was shown to occur over the same concentration range (>100 nM), regardless of tryptophan concentration, whereas binding to sequences bearing partial specificity ratio between 100 and 1000, depending upon the salt concentration. Even in absence of added KCl, the specificity ratio of trp repressor was greater than 100, implicating a significant free energy contribution from non-electrostatic interaction forces.

Bacterial Proteins↗
PubMed1997

MyoD-E12 heterodimers and MyoD-MyoD homodimers are equally stable.

Muscle development is controlled by the MyoD family of basic helix-loop-helix (bHLH) DNA-binding proteins. These proteins dimerize with ubiquitous products of the E2A gene (E12 and E47) and bind in a sequence-specific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been utilized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociation (deltaG) and the equilibrium dissociation constants (K(D)) for the protein-protein interactions are reported. The deltaG for the MyoD homodimers in 100 mM KCl was 8.7 kcal/mol (K(D) = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigorous confidence limit testing. The titrations of E12-dansyl with MyoD yielded a free energy of 8.3 kcal/mol with tighter confidence limits, +0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers and MyoD-E12 heterodimers are relatively weak complexes of approximately the same stability. E12 does not form stable homo-oligomeric complexes; remaining monomeric at concentrations as high as 20 microM. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterodimers versus MyoD homodimers with DNA binding sites is not due to preferential heterodimerization.

Animals↗
PubMed1997

The kinetic basis for the stabilization of staphylococcal nuclease by xylose.

The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence-detected pressure-jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234-10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909-4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate-limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.

Enzyme Stability↗
PubMed1996

Characterization of charge change super-repressor mutants of trp repressor: effects on oligomerization conformation, ligation and stability.

We have carried out a physical characterization of mutant repressor proteins of the trp repressor system of Escherichia coli by circular dichroism, chemical denaturation, and 8-anilino-1-naphthalenesulfonate binding. We have also probed the protein-protein interactions via fluorescence anisotropy and lifetime measurements and measured the thermodynamics of ligand (L-tryptophan) binding by isothermal titration calorimetry. Here, we present investigations of four charge change super-repressor mutants: EK13, EK18, DN46 and EK49, and compare these results with those previously obtained for wild-type trp repressor and the AV77 super-repressor mutant. These studies demonstrate that super-repressor phenotypes may result from changes in operator affinity (DN46, EK49), protein-protein interactions (EK18), as well as the coupling of folding to ligand binding (AV77, EK13, EK18). Correlations between the oligomerization behavior and cooperativity of DNA binding for some of these mutants indicate that coupling of oligomerization to DNA binding modulates operator site occupation giving rise to the super-repressor phenotype. The present results underscore the complex interplay between the multiple equilibria in this system. Moreover, they provide insights into the structural basis for the mutational perturbation of the energetics of this classical allosterically controlled transcriptional regulator.

Anilino Naphthalenesulfonates↗
PubMed1996

Testing the correlation between delta A and delta V of protein unfolding using m value mutants of staphylococcal nuclease.

The application of hydrostatic pressure to aqueous protein solutions results in the unfolding of the protein structure because the protein-solvent system volume is smaller for the unfolded state. Contributions to this decrease in volume upon unfolding (delta Vu) derive from altered interactions of the protein with solvent and are presumed to include electrostriction of charged residues, elimination of packing defects, and hydration of hydrophobic surfaces upon unfolding. If the contribution of hydrophobic surface area solvation to the observed volume change of unfolding were large and negative, as is generally assumed, then one would expect to find a correlation between the amount of surface area exposed on unfolding, delta A(u), and the volume change, delta Vu. In order to test this correlation, we have determined delta Vu for two mutants of staphylococcal nuclease, A69T + A90S and H121P, whose unfolding by denaturant is, respectively, either significantly more (28%) or significantly less (28%) cooperative than that observed for wild-type (WT). This cooperativity coefficient or m value has been shown to correlate with delta A(u). If, in turn, delta Vu is correlated with delta A(u), we would expect the m+ mutant, A69T + A90S, to exhibit a delta Vu that is more negative than WT nuclease, while the delta Vu for the m- mutant, H121P, should be smaller in absolute value. To verify the correlation between m value and delta A(u) for these mutants, we determined the xylose concentration dependence of the stability of each mutant at atmospheric pressure and as a function of pressure. The efficiency of xylose stabilization was found to be much greater for the m+ mutant than for WT, consistent with an increase in delta A(u), while that of the m- mutant was found to be only slightly greater than for WT, indicating that other factors may contribute to the denaturant m value in this case. Regardless of the denaturant m value or the effect of xylose on stability, the volume changes upon unfolding for both mutants were found to be within error of that observed for WT. Thus, there does not appear to be a correlation between the volume change and the change in exposed surface area upon unfolding. We have previously shown a lack of pH dependence of the volume change, ruling out electrostriction as a dominant contribution to delta Vu of nuclease. These studies implicate either compensation between polar and nonpolar hydration or excluded volume effects as the major determinant for the value of delta Vu.

Chromatography, Gel↗
PubMed1996

High-pressure denaturation of staphylococcal nuclease proline-to-glycine substitution mutants.

Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to-glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42.

Enzyme Stability↗