Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.     

Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations. Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.     

The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366-14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co(2+)/Co(2+)-PTE. This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9-10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry-Eyring model. A temperature-induced conformational transition (T(m) approximately equal to 58 degrees C) and an early growing of aggregates were observed. Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T(m) was neither native nor active. Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76 degrees C. Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T approximately equal to 45 degrees C and an inactive unfolded state populated at T approximately equal to 58 degrees C, which leads to denatured aggregates. Thus, the wild type Co(2+)/Co(2+)-PTE displays a middle-range thermostability. Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.     

Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy

A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated ‘invisible’ protein states that exchange with a ‘visible’ ground state on the millisecond time-scale. The utility of the approach is demonstrated with an application to an I58D mutant of the Pfl6 Cro protein that undergoes exchange between the native, folded state and a cold denatured, unfolded conformational ensemble that is populated at a level of 6% at 2.5°C. A wide distribution of amide temperature coefficients is measured for the unfolded state. The distribution is centered about –5.6 ppb/K, consistent with an absence of intra-molecular hydrogen bonds, on average. However, the large range of values (standard deviation of 2.1 ppb/K) strongly supports the notion that the unfolded state of the protein is not a true random coil polypeptide chain.     

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.     

Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter V(max) and K(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.     

Mutational analysis of NADH-binding residues in triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P

Triphenylmethane reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved GXXGXXG motif near its N-terminus, which corresponds to a conserved structural motif of known dinucleotide-binding proteins. To verify whether some of these glycine residues are important for the enzyme catalysis, these three glycine residues (Gly-7, Gly-10 and Gly-13) were individually replaced by alanine using site-directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences as compared with the wild type. The V(max)/K(m) values of mutants G7A and G13A for both Basic fuchsin and NADH were increased about three and twofold over that of the wild type, respectively, whereas the V(max)/K(m) value of mutant G10A were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate and cofactor for the catalytic activity of TMR.     

A fragment of staphylococcal nuclease with an OB-fold structure shows hydrogen-exchange protection factors in the range reported for "molten globules".

Hydrogen-exchange rates for an OB-fold subdomain fragment of staphylococcal nuclease have been measured at pH 4.7 and 4 degrees C, conditions close to the minimum of acid/base catalyzed exchange. The strongest protection from solvent exchange is observed for residues from a five-stranded beta-barrel in the NMR structure of the protein. Protection factors, calculated from the experimental hydrogen-exchange rates, range between 1 and 190. Similarly small protection factors have in many cases been attributed to "molten globule" conformations that are supposed to lack a specific tertiary structure. The present results suggest that marginal protection from solvent exchange does not exclude well-defined structure.     

Acid-Induced Formation of Molten Globule States in the Wild Type <Emphasis Type="Italic">Escherichia coli</Emphasis> 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A,A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.     

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

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

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

The unfolded state of NTL9 is compact in the absence of denaturant

Interest in the unfolded state of proteins has grown with the realization that this state can have considerable structure in the absence of denaturants. Natively unfolded proteins, mutations that unfold proteins under native conditions, and changes in pH that induce unfolding are attractive models for the unfolded state in the absence of denaturant. The unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) was previously shown to contain significant non-native electrostatic interactions [Cho, J. H., Sato, S., and Raleigh, D. P. (2004) J. Mol. Biol. 338, 827-837]. NTL9 has a mixed alpha-beta structure and folds via a two-state mechanism. We have generated a model of the unfolded state of NTL9 in the absence of denaturant by substitution of an alanine for phenylalanine 5 located in the core of this protein. The CD spectrum of the variant, denoted as F5A, exhibits significantly less structure than the wild type; however, the mean residue ellipticity of F5A at 222 nm (-8200 deg cm(2) dmol(-)(1)) is considerably larger than expected for a fully unfolded protein, indicating that residual secondary structure is populated. F5A also has more residual structure than the urea-unfolded wild type. The stability of F5A is estimated to be at least 1 kcal/mol unfavorable, showing that the unfolded state is populated to 84% or more. NMR pulsed-field gradient measurements yield a hydrodynamic radius of 16.1 A for wild-type NTL9 and 20.8 A for the F5A variant in native buffer. The physiologically relevant unfolded state of wild-type NTL9 is likely to be even more compact than F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant. The hydrodynamic radius of F5A increases to 25.9 A in 8 M urea, and a value of 23.5 A is obtained for the wild type under similar conditions. The results show that the unfolded state of F5A in the absence of denaturant is more compact and contains more structure than the urea-unfolded form.     

Kinetic analysis of molecular dynamics simulations reveals changes in the denatured state and switch of folding pathways upon single-point mutation of a beta-sheet miniprotein

The effects of a single-point mutation on folding thermodynamics and kinetics are usually interpreted by focusing on the native structure and the transition state. Here, the entire conformational spaces of a 20-residue three-stranded antiparallel beta-sheet peptide (double hairpin) and of its single-point mutant W10V are sampled close to the melting temperature by equilibrium folding-unfolding molecular dynamics simulations for a total of 40 micros. The folded state as well as the most populated free energy basins in the denatured state are isolated by grouping conformations according to fast relaxation at equilibrium. Such kinetic analysis provides more detailed and useful information than a simple projection of the free energy. The W10V mutant has the same native structure as the wild type peptide, and similar folding rate and stability. In the denatured state, the N-terminal hairpin is about 20% more structured in W10V than the wild type mainly because of van der Waals interactions. Notably, the W10V mutation influences also the van der Waals energy at the transition state ensemble causing a shift in the ratio of fluxes between two different transition state regions on parallel folding pathways corresponding to nucleation at either of the two beta-hairpins. Previous experimental studies have focused on the effects of denaturant-dependent or temperature-dependent changes in the structure of the denatured state. The atomistic simulations show that a single-point mutation in the central strand of a beta-sheet peptide results in remarkable changes in the topography of the denatured state ensemble. These changes modulate the relative accessibility of parallel folding pathways because of kinetic partitioning of the denatured state. Therefore, the observed dependence of the folding process on the starting ensemble raises questions on the biological significance of in vitro folding studies under strongly denaturing conditions.     

Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.     

NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.     

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.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.