Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Robustness of the long-range structure in denatured staphylococcal nuclease to changes in amino acid sequence

A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.     

A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease.

In an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortle D, 1995, Biochemistry 34:15895-15905), we reported evidence of a three-strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local "non-native" interaction with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, this unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data that confirm the important role of interactions involving four hydrophobic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the structure formed by the chain segments corresponding to beta 1-beta 2-beta 3-h, interactions that are not present in the native state. Glycine substitutions for each of these large hydrophobic residues destabilizes or disrupts this beta structure, as assessed by HN line sharpening and changes in the CD spectrum. The 13C resonances of the carbonyl carbon for several of the residues in this structure indicate conformational dynamics that respond in a complex way to addition of urea or changes in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native-like three-strand beta sheet. Instead, the data are more consistent with the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by hydrophobic interactions.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state

The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (Delta), GdnHCl (Delta), and heat (Delta). Second, by numerical integration of H(+) titration curves (Delta) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by Delta, Delta, and Delta was comparable but significantly different from the one described by Delta. The decrease in Delta between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in Delta, Delta, and Delta in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, Delta represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by Delta and by Delta between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D). These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in Delta and Delta at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.     

Compact denatured state of a staphylococcal nuclease mutant by guanidinium as determined by resonance energy transfer.

The protein from a mutant clone of staphylococcal nuclease with a cysteine substituting for a lysine at position 78 was prepared and labeled with a cysteine-specific fluorescent probe 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). Time-resolved nonradiative energy-transfer studies were done using the single tryptophan at position 140 as the energy donor and the IAEDANS as the receptor. Changes in distance and distance distributions were observed as a function of increasing guanidinium (GuHCl) concentration (0-2 M) and in the presence or absence of Ca2+ and inhibitor 2'-deoxythymidine 3',5'-diphosphate (pdTp). In the native state, both the ternary complex and the noncomplexed protein are best fit with one population having an average donor-acceptor distance of approximately 23 A and an "apparent" full width at half-maximum (fwhm) of distance distribution of approximately 18 A. Besides the contribution of linker arm of the acceptor, it appears that there are some conformational heterogeneties either due to the disordering of the tryptophan region or due to the whole protein in the native state. During GuHCl unfolding, the average distance remains relatively constant up to GuHCl concentrations where both the ternary complex and the ligand-free protein are denatured (1-1.3 M). The compact denatured states persist up to 2 M GuHCl. At 2 M GuHCl, the heterogeneity of the denatured state in the ternary complex is much larger than that of the ligand-free nuclease. The results show that the denatured states of staphylococcal nuclease mutant K78C by GuHCl are compact and these compact denatured states are likely due to residual structures or incompletely disrupted hydrophobic cores under these conditions.     

Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR

The self-association reaction of denatured staphylococcal nuclease fragments, urea-denatured G88W110, containing residues 1-110 and mutation G88W, and physiologically denatured 131-residue Delta 131 Delta, have been characterized by NMR at close to neutral pH. The two fragments differ in the extent and degree of association due to the different sequence and experimental conditions. Residues 13-39, which show significant exchange line broadening, constitute the main association interface in both fragments. A second weak association region was identified involving residues 79-105 only in the case of urea-denatured G88W110. For residues involved in the association reaction, significant suppression of the line broadening and small but systematic chemical shift variation of the amide protons were observed as the protein concentration decreased. The direction of chemical shift change suggests that the associated state adopts mainly beta-sheet-like conformation, and the beta-hairpin formed by strands beta 2 and beta 3 is native-like. The apparent molecular size obtained by diffusion coefficient measurements shows a weak degree of association for Delta 131 Delta below 0.4 mM protein concentration and for G88W110 in 4 M urea. In both cases the fragments are predominantly in the monomeric state. However, the weak association reaction can significantly influence the transverse relaxation of residues involved in the association reaction. The degree of association abruptly increases for Delta 131 Delta above 0.4 mM concentration, and it is estimated to form a 4 to 8 mer at 2 mM. It is proposed that the main region involved in association forms the core structure, with the remainder of residues largely disordered in the associated state. Despite the obvious influence of the association reaction on the slow motion of the backbone, the restricted mobility on the nanosecond timescale around the region of strand beta 5 is essentially unaffected by the association reaction and degree of denaturation.     

Electrostatic effects in unfolded staphylococcal nuclease

Structure-based calculations of pKa values and electrostatic free energies of proteins assume that electrostatic effects in the unfolded state are negligible. In light of experimental evidence showing that this assumption is invalid for many proteins, and with increasing awareness that the unfolded state is more structured and compact than previously thought, a detailed examination of electrostatic effects in unfolded proteins is warranted. Here we address this issue with structure-based calculations of electrostatic interactions in unfolded staphylococcal nuclease. The approach involves the generation of ensembles of structures representing the unfolded state, and calculation of Coulomb energies to Boltzmann weight the unfolded state ensembles. Four different structural models of the unfolded state were tested. Experimental proton binding data measured with a variant of nuclease that is unfolded under native conditions were used to establish the validity of the calculations. These calculations suggest that weak Coulomb interactions are an unavoidable property of unfolded proteins. At neutral pH, the interactions are too weak to organize the unfolded state; however, at extreme pH values, where the protein has a significant net charge, the combined action of a large number of weak repulsive interactions can lead to the expansion of the unfolded state. The calculated pKa values of ionizable groups in the unfolded state are similar but not identical to the values in small peptides in water. These studies suggest that the accuracy of structure-based calculations of electrostatic contributions to stability cannot be improved unless electrostatic effects in the unfolded state are calculated explicitly.     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

Thermodynamics of staphylococcal nuclease denaturation. I. The acid-denatured state.

Using high-sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower delta Cp and delta H for the denaturation reaction. The acid-denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid-denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of delta H on pH.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.     

Interactions between subdomains in the partially folded state of staphylococcal nuclease

Staphylococcal nuclease can be roughly divided into a beta-subdomain in N-terminal and an alpha-subdomain in C-terminal. They fold sequentially under certain conditions, causing a partially folded intermediate state in which the native-like beta-barrel persists while alpha-helix regions largely disorder. To investigate the possible long-range interactions between the two subdomains in the intermediate, N-terminal fragments have been used as intermediate analogues, with polypeptide ending at positions 102, 110, 121 and 135 and with a tryptophan substitution at position 66 or 88 to facilitate the observation of the beta-barrel. Segment-resolved interactions between beta-barrel and residues 103-135 were identified by comparing their spectroscopic properties of fluorescence, circular dichroism and NMR and by their stability. Except for unstable V66W102, the guanidine and thermal denaturation of fragments are cooperative and well approximated by the two-state transition. Minimal stable structure units of both tryptophan-containing fragments comprise residues 1-110. With the main interaction in segment 103-135, residues 103-110 contribute approximate 2 kcal/mol to the stability. Elongation of C-terminal from 110 residue neither increases the stability nor alters the structure core of the G88W fragments. However, residues 111-121 influence the tertiary structure of the V66W fragments suggesting its minor interactions with beta-barrel.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Characterization of the residual structure in the unfolded state of the Delta131Delta fragment of staphylococcal nuclease

The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular dynamics simulations to characterise the residual structure of the Delta131Delta fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that Delta131Delta under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild-type staphylococcal nuclease. Only rarely, however, all these interactions are simultaneously realized to generate conformations with an overall native topology.     

Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions

In an attempt to develop a model of the denatured state of staphylococcal nuclease that can be analyzed experimentally under physiological conditions, a series of four large fragments of this small protein which extend from residues 1 to 103, 1 to 112, 1 to 128, and 1 to 136 have been generated through the overexpression of nuclease genes containing stop codons at defined positions. Large amounts of protein fragments were accumulated in induced cells and were purified by carrying out all fractionation steps in the presence of 6 M urea. The far-ultraviolet circular dichroism spectra of all four fragments suggested the presence of small to moderate amounts of residual structure. When the CD spectra were monitored as a function of concentrations of the tight-binding ligands Ca2+ and thymidine 3',5'-bisphosphate and the known affinity constants for wild-type nuclease (1-149) were used, apparent equilibrium constants of 160 and 2000 for the reversible denaturation reaction for fragments 1-136 and 1-128, respectively, were estimated. Four single and two double mutations, all of which exhibit unusual behavior in the full-length protein on solvent denaturation [Shortle, D., & Meeker, A. K. (1986) Proteins: Struct., Funct., Genet. 1, 81-89] and thermal denaturation [Shortle, D., Meeker, A. K., & Freire, E. (1988) Biochemistry 27, 4761-4768], were recombined into the 1-136 and 1-128 fragment expression vectors, and purified mutant fragments were characterized.(ABSTRACT TRUNCATED AT 250 WORDS)