Analysis of electrostatic interactions in the denatured state ensemble of the N-terminal domain of L9 under native conditions

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.     

Residual electrostatic effects in the unfolded state of the N-terminal domain of L9 can be attributed to nonspecific nonlocal charge-charge interactions

Residual electrostatic interactions in the unfolded state of the N-terminal domain of L9 (NTL9) were found by Kuhlman et al. [(1999) Biochemistry 38, 4896-4903]. These residual interactions are analyzed here by the Gaussian-chain model [Zhou, H.-X. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3569-3574]. The original model is made more realistic by replacing "standard" model-compound pK(a) values for ionizable groups by those measured by Kuhlman et al. in peptide fragments of NTL9. The predicted pH dependence of the unfolding free energy is in agreement with experiment over the pH range of 1-7 at ionic strengths of 100 and 750 mM. This indicates that the residual electrostatic effects in the unfolded state of NTL9 can be attributed to nonspecific nonlocal charge-charge interactions.     

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.     

pH-dependent interactions and the stability and folding kinetics of the N-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding

The role of electrostatic interactions in the stability and the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) was investigated by determining the effects of varying the pH conditions. Urea denaturations and thermal unfolding experiments were used to measure the free energy of folding, DeltaG degrees, at 18 different pH values, ranging from pH 1.1 to pH 10.5. Folding rates were measured at 19 pH values between pH 2.1 and pH 9.5, and unfolding rates were determined at 15 pH values in this range using stopped-flow fluorescence experiments. The protein is maximally stable between pH 5.5 and 7.5 with a value of DeltaG degrees =4.45 kcal mol(-1). The folding rate reaches a maximum at pH 5.5, however the change in folding rates with pH is relatively modest. Over the pH range of 2.1 to 5.5 there is a small increase in folding rates, ln (k(f)) changes from 5.1 to 6.8. However, the change in stability is more dramatic, with a difference of 2.6 kcal mol(-1) between pH 2.0 and pH 5.4. The change in stability is largely due to the smaller barrier for unfolding at low pH values. The natural log of the unfolding rates varies by approximately four units between pH 2.1 and pH 5.5. The stability of the protein decreases above pH 7.5 and again the change is largely due to changes in the unfolding rate. ln (k(f)) varies by less than one unit between pH 5.5 and pH 9.5 while DeltaG degrees decreases by 2.4 kcal mol(-1) over the range of pH 5. 4 to pH 10.0, which corresponds to a change in ln K(eq) of 4.0. These studies show that pH-dependent interactions contribute significantly to the overall stability of the protein but have only a small effect upon the folding kinetics, indicating that electrostatic interactions are weakly formed in the transition state for folding.     

Thermodynamics and kinetics of non-native interactions in protein folding: a single point mutant significantly stabilizes the N-terminal domain of L9 by modulating non-native interactions in the denatured state

Comparatively little is known about the role of non-native interactions in protein folding and their role in both folding and stability is controversial. We demonstrate that non-native electrostatic interactions involving specific residues in the denatured state can have a significant effect upon protein stability and can persist in the transition state for folding. Mutation of a single surface exposed residue, Lys12 to Met, in the N-terminal domain of the ribosomal protein L9 (NTL9), significantly increased the stability of the protein and led to faster folding. Structural and energetic studies of the wild-type and K12M mutant show that the 1.9 kcal mol(-1) increase in stability is not due to native state effects, but rather is caused by modulation of specific non-native electrostatic interactions in the denatured state. pH dependent stability measurements confirm that the increased stability of the K12M is due to the elimination of favorable non-native interactions in the denatured state. Kinetic studies show that the non-native electrostatic interactions involving K12 persist in the transition state. The analysis demonstrates that canonical Phi-values can arise from the disruption of non-native interactions as well as from the development of native interactions.     

Characterization of large peptide fragments derived from the N-terminal domain of the ribosomal protein L9: definition of the minimum folding motif and characterization of local electrostatic interactions

A set of peptides derived from the N-terminal domain of the ribosomal protein L9 (NTL9) have been characterized in an effort to define the minimum unit of this domain required to fold and to provide model peptides for the analysis of electrostatic interactions in the unfolded state. NTL9 is a 56-residue alpha-beta protein with a beta1-loop-beta2-alpha1-beta3-alpha2 topology. The beta-sheet together with the first helix comprise a simple example of a common supersecondary motif called the split beta-alpha-beta fold. Peptides corresponding to the beta1-loop-beta2 unit are unstructured even when constrained by an introduced disulfide. The pK(a)s of Asp-8 and Glu-17 in these peptides are slightly lower than the values found for shorter peptides but are considerably higher than the values in NTL9. A 34-residue peptide, which represents the beta1-loop-beta2-alpha1 portion of NTL9, is also unstructured. In contrast, a 39-residue peptide corresponding to the entire split beta-alpha-beta motif is folded and monomeric as judged by near- and far-UV CD, two-dimensional NMR, ANS binding experiments, pK(a) measurements, and analytical ultracentrifugation. The fold is very similar to the structure of this region in the intact protein. Thermal and urea unfolding experiments show that it is cooperatively folded with a DeltaG degrees of unfolding of 1.8-2.0 kcal/mol and a T(m) of 58 degrees C. This peptide represents the first demonstration of the independent folding of an isolated split beta-alpha-beta motif, and is one of only four naturally occurring sequences of fewer than 40 residues that has been shown to fold cooperatively in the absence of disulfides or ligand binding.     

The cold denatured state is compact but expands at low temperatures: hydrodynamic properties of the cold denatured state of the C-terminal domain of L9

A point mutation of a small globular protein, the C-terminal domain of L9 destabilizes the protein and leads to observable cold-denaturation at temperatures above zero. The cold denatured state is in slow exchange with the native state on the NMR time scale, and this allows the hydrodynamic properties of the cold unfolded state and the native state to be measured under identical conditions using pulsed-field gradient NMR diffusion measurements. This provides the first experimental measurement of the hydrodynamic properties of a cold unfolded protein and its folded form under identical conditions. Hydrodynamic radii of the cold-induced unfolded states were measured for a set of temperatures ranging from 2 °C to 25 °C at pD 6.6 in the absence of denaturant. The cold unfolded state is compact compared to the urea or acid unfolded state and a trend of increasing radii of hydration is observed as the temperature is lowered. These observations are confirmed by experiments on the same protein at pD 8.0, where it is more stable, in the presence of a modest concentration of urea. The expansion of the cold-denatured state at lower temperatures is consistent with the temperature dependence of hydrophobic interactions.     

pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure

It is now recognized that unfolded states of globular proteins are not random coils but instead can contain significant amounts of residual structure. Here, we combine amide H/D exchange studies and thermodynamic measurements to probe pH dependent structure in the unfolded state of the small, mixed alpha-beta protein CTL9. The m value measured by urea denaturation is strongly dependent upon pD, increasing by 40% from pD 7.5 to 4.85. Likewise, the change in heat capacity upon unfolding, deltaCp(o), increases significantly from pD 7.5 to 5.5. These studies argue that the unfolded state contains interactions, presumably hydrophobic in nature, that lead to a more compact state at high pH. The expansion at lower pH correlates with the estimated unfolded state pKa values of the three histidines in CTL9 with additional contributions from acid side chains at the lower pH. Amide H/D exchange studies were conducted at pD 5.0, 6.0, and 7.0. At pD 5.0, the exchange rates could be measured for 44 residues, 29 of which exchanged by global unfolding. No evidence was found for any super protected sites, that is, sites that exchange at rates slower than those expected for global exchange. The estimated precision for the experiments limits detection to residues that are protected 2.3-fold above the intrinsic exchange rate. Thirty-seven residues could be followed at pD 6 and 27 residues at pD 7. Again no evidence for a significant super protected structure was observed. The properties of CTL9(11) are compared to other structured denatured states.     

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.     

Electrostatic interactions in the denatured state ensemble: their effect upon protein folding and protein stability

It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well as hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol^-1 to the stability of the DSE.     

Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins

The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble.     

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: comparison with the intact protein.

Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1-56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N-terminal domain are very similar to those previously measured for the corresponding region in the full-length protein (Lillemoen J et al., 1997, J Mol Biol 268:482-493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N-terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.     

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

Contact order dependent protein folding rates: kinetic consequences of a cooperative interplay between favorable nonlocal interactions and local conformational preferences

Physical mechanisms underlying the empirical correlation between relative contact order (CO) and folding rate among naturally occurring small single-domain proteins are investigated by evaluating postulated interaction schemes for a set of three-dimensional 27mer lattice protein models with 97 different CO values. Many-body interactions are constructed such that contact energies become more favorable when short chain segments sequentially adjacent to the contacting residues adopt native-like conformations. At a given interaction strength, this scheme leads to folding rates that are logarithmically well correlated with CO (correlation coefficient r = 0.914) and span more than 2.5 orders of magnitude, whereas folding rates of the corresponding Gō models with additive contact energies have much less logarithmic correlation with CO and span only approximately one order of magnitude. The present protein chain models also exhibit calorimetric cooperativity and linear chevron plots similar to that observed experimentally for proteins with apparent simple two-state folding/unfolding kinetics. Thus, our findings suggest that CO-dependent folding rates of real proteins may arise partly from a significant positive coupling between nonlocal contact favorabilities and local conformational preferences.     

Histidine-aromatic interactions in barnase. Elevation of histidine pKa and contribution to protein stability.

Aromatic side-chains are found in the vicinity of histidine residues in many proteins and protein complexes. We have studied the interaction between a histidine residue (His18) and aromatic residues at position 94 in barnase. Three different techniques have been applied to show that Trp94 interacts more strongly with the protonated form of His18. The aromatic-histidine interaction stabilizes the protonated form of histidine by 0.8 to 1 kcal mol-1 relative to the unprotonated and, thereby, increases its pKa value. This was shown indirectly from the pH dependence of the stability of the wild-type protein and the mutant Trp94----Leu; and directly from the difference in pKa of His18 between wild-type barnase and the same mutant protein, and from double-mutant cycles that measure the total interaction energy of Trp94 with His18 at both low and high pH. When Trp94 is replaced by other aromatic amino acids, the strength of the interaction decreases in the series His-Trp greater than His-Tyr greater than His-Phe. The interaction is not masked by high salt concentrations. The raising of the pKa value of His18 by interaction with Trp94 is shown to be consistent with solution studies with model compounds. The histidine-aromatic interaction could have implications in binding and catalysis for modulation of the histidine pKa value.     

Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.     

Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (phi value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small alpha-beta protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T(m) and Delta G(0) of unfolding. The folding rate also decreased. The parameter Phi(H/D), defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D(2)O background and 0.75 in a H(2)O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the beta-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.     

Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of mu-conotoxin GIIIA (muCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of muCTX only partially block the single-channel current, enabling us to directly monitor properties of both muCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2-3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than -100 mV at the level of the "outer charged ring" formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand--receptor complex during individual molecular transitions and defined conformational states.     

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.