Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin.

Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein.     

Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids

Pyrococcus furiosus (Pf) rubredoxin is the most thermostable protein characterized to date. Reflecting the complications arising from irreversible denaturation of this protein, predictions of which structural regions confer differential thermal stability have utilized kinetic stability measurements, hydrogen exchange protection factors, long range hydrogen bond NMR spin couplings, and molecular dynamics simulations, and have primarily implicated the three-stranded beta-sheet and the adjacent metal binding site. Herein, NMR chemical exchange experiments demonstrate reversible two-state unfolding at the thermal transition temperature (T(m)) for hybrids of Pf and the mesophile Clostridium pasteurianum (Cp) rubredoxins which interchange residues 14-33, the so-called multi-turn segment. This complementary pair of hybrid rubredoxins exhibits largely additive incremental thermal stabilizations vs. the parental proteins. Both stabilization free energy measurements as well as incremental T(m) values indicate that a minimum of 37% of the total differential thermal stability resides in this multi-turn segment. Such a proportionality between DeltaDeltaG and incremental T(m) values is predicted for hybrid pairs exhibiting thermodynamic additivity in which the differential stability is predominantly enthalpic.     

Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins

The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.     

Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation

The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C. This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data.     

Tetrathiolate coordination of germanium(IV) in a protein active site

The tetracysteine metal coordination site of the rubredoxins from Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) are shown to stably bind the inorganic Ge(IV) ion. This is the first characterized coordination complex of tetravalent germanium with a biological macromolecule. Zn(II), Ga(III) and Ge(IV) substitution yields differential NMR chemical shifts for the 1H and 15N amide resonances throughout much of the protein structure. The differential shifts for the six backbone amides that hydrogen bond to the metal-coordinated sulfurs indicate that the pseudo 2-fold symmetry of the active site is more closely maintained in the hyperthermophile Pf rubredoxin than in its mesophile Cp homolog. These three metal substitutions form an isoelectronic series of small diamagnetic proteins for which reference structures are known to 1A resolution. These series provide a promising system to analyze theoretical predictions of the effects of differential charge distribution on chemical shifts from both proximal and long range interactions.     

2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: contributions of the beta-sheet to thermostability.

Based on 2D 1H-1H and 2D and 3D 1H-15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd). The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the three-stranded beta-sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the beta-sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci 1:1508-1521], the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the beta-sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.     

Additivity in both thermodynamic stability and thermal transition temperature for rubredoxin chimeras via hybrid native partitioning

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.     

NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin

Background  

Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions.     

Thermodynamic basis for the increased thermostability of CheY from the hyperthermophile Thermotoga maritima.

The CheY protein isolated from the hyperthermophile Thermotoga maritima is much more resistant to thermally induced unfolding than is its counterpart from the mesophile Bacillus subtilis. To determine the basis of this increased thermostability, the temperature dependence of the free energy of unfolding was determined for these CheY homologues using denaturant-induced unfolding experiments. This allowed comparison of T. maritima CheY with B. subtilis CheY and determination of the thermodynamic qualities responsible for the enhanced thermostability of T. maritima CheY. The stability of the thermophilic CheY protein is a direct result of the increased enthalpy contribution at the temperature of zero entropy, T(s), and the decreased heat capacity change upon unfolding, resulting in a decreased dependence of the free energy of unfolding on temperature. It was found that neither purely entropic nor purely enthalpic contributions alone (as reflected by T(s)) were sufficient to account for the increase in stability.     

Molecular dynamics study of a hyperthermophilic and a mesophilic rubredoxin

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.     

Role of native-state structure in rubredoxin native-state hydrogen exchange

Amide exchange rates were measured for Pyrococcus furiosus (Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 A, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions in Pf rubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.     

Investigations of the thermostability of rubredoxin models using molecular dynamics simulations.

The affects of differences in amino acid sequence on the temperature stability of the three-dimensional structure of the small beta-sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100 degrees C, were compared to three mesophilic Rds of known X-ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature-dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature-dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.     

The role of backbone stability near Ala44 in the high reduction potential class of rubredoxins

Rubredoxins may be separated into high and low reduction potential classes, with reduction potentials differing by approximately 50 mV. Our previous work showed that a local shift in the polar backbone due to an A(44) versus V(44) side-chain size causes this reduction potential difference. However, this work also indicated that in the low potential Clostridium pasteurianum (Cp) rubredoxin, a V(44) --> A(44) mutation causes larger local backbone flexibility, because the V(44) side-chain present in the wild-type (wt) is no longer present to interlock with neighboring residues to stabilize the subsequent G(45). Since Pyrococcus furiosus (Pf) and other high potential rubredoxins generally have a P(45), it was presumed that a G(45) --> P(45) mutation might stabilize a V(44) --> A(44) mutation in Cp rubredoxin. Here crystal structure analysis, energy minimization, and molecular dynamics (MD) were performed for wt V(44)G(45), single mutant A(44)G(45) and double mutant A(44)P(45) Cp, and for wt A(44)P(45) Pf rubredoxins. The local structural, dynamical, and electrostatic properties of Cp gradually approach wt Pf in the order wt Cp to single to double mutant because of greater sequence similarity, as expected. The double mutant A(44)P(45) Cp exhibits increased backbone stability near residue 44 and thus enhances the probability that the backbone dipoles point toward the redox site, which favors an increase in the electrostatic contribution to the reduction potential. It appears that the electrostatic potential of residue 44 and the solvent accessibility to the redox are both determinants for the reduction potentials of homologous rubredoxins. Overall, these results indicate that an A(44) in a rubredoxin may require a P(45) for backbone stability whereas a V(44) can accommodate a G(45), since the valine side-chain can interlock with its neighbors.     

Thermal stability of the [Fe(SCys)4] site in Clostridium pasteurianum rubredoxin: contributions of the local environment and Cys ligand protonation

Thermal denaturation of the mesophilic rubredoxin from Clostridium pasteurianum occurs through a number of temperature-dependent steps, the last and irreversible one being release of iron from the [Fe(2+)(SCys)(4)] site. We show here that thermally induced [Fe(2+)(SCys)(4)] site destruction is largely determined by the local environment, and not directly connected to thermostability of the native polypeptide fold of rubredoxin. Hydrophobic residues on the protein surface, V8 and L41, that shield the [Fe(SCys)(4)] site from solvent and form N-H(.)S hydrogen bonds to the metal-coordinating sulfurs, were mutated to residues with both uncharged and charged side chains. On these mutated rubredoxins the temperature dependence was measured for: (1) global unfolding of the protein by NMR, (2) loss of Fe(2+)at various ionic strengths and pH values, (3) the rates of non-denaturing displacement of Fe(2+) by Cd(2+) or Zn(2+). For reversible temperature-dependent changes in the global protein folding that occur prior to loss of iron, no thermostability differences were found among the wild-type, V8A, V8D, L41R, and L41D rubredoxins. However, for irreversible loss of iron from the [Fe(2+)(SCys)(4)] site, relative to the wild-type protein, L41R was more thermostable, V8A was somewhat less thermostable, and the acidic mutants L41D, V8D and [V8D, L41D] showed dramatically lowered thermostability. Lower pH facilitated - both kinetically and thermodynamically - thermally induced iron release, likely through protonation of ligand cysteines' thiols. For all of the rubredoxins a direct correlation was found between the midpoint temperature for thermally induced Fe(2+) loss and the rate of non-denaturing Fe(2+) displacement by Cd(2+) or Zn(2+) at room temperature. A mechanism is proposed involving transient movement of residue-8 and -41 side chains, allowing, and, in the case of negatively charged side chains, also facilitating, attack of a ligand cysteine by the incoming positively charged species (H(+), Cd(2+), or Zn(2+)). Thus, localized charge density and solvent accessibility modulate the stability of Fe(2+) ligation in rubredoxin. However, the reduced [Fe(SCys)(4)] site does not control the thermostability of the native polypeptide fold of rubredoxin.     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.     

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.     

Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.     

Mutational analysis of differences in thermostability between histones from mesophilic and hyperthermophilic archaea

Amino acid residues responsible for the large difference in thermostability between HMfB and HFoB, archaeal histones from the hyperthermophile Methanothermus fervidus and the mesophile Methanobacterium formicicum, respectively, have been identified by site-specific mutagenesis. The thermal denaturation of approximately 70 archaeal histone variants has been monitored by circular dichroism, and the data generated were fit to a two-state unfolding model (dimer-->two random coil monomers) to obtain a standard-state (1M) melting temperature for each variant dimer. The results of single-, double-, and triple-residue substitutions reveal that the much higher stability of rHMfB dimers, relative to rHFoB dimers, is conferred predominantly by improved intermolecular hydrophobic interactions near the center of the histone dimer core and by additional favorable ion pairs on the dimer surface.     

Contribution of the [Fe<Superscript>II</Superscript>(SCys)<Subscript>4</Subscript>] site to the thermostability of rubredoxins

The thermostabilities of Fe²⁺ ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH...S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)₄] site, and substitutions at this location affect the redox properties of the [Fe(SCys)₄] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V exchange mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)₄] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn²⁺, Cd²⁺) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and ¹H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe^II(SCys)₄] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe^II(SCys)₄] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)₄] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H⁺, Cd²⁺) on the Cys42 sulfur ligand at the [Fe(SCys)₄] site occurs through the V8 side and not the V44 side of the iron site.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0525-4Abbreviations BPS bathophenanthroline sulfonate, sodium salt - Cp Rd (Pf Rd, Dv Rd) recombinant rubredoxin from Clostridium pasteurianum (Pyrococcus furiosus, Desulfovibrio vulgaris) - HEPES hydroxyethylpiperazineethanesulfonic acid - MES morpholinoethanesulfonic acid - Tris tris(hydroxymethyl)aminomethane - wt wild-type - ZnRd recombinant rubredoxin containing a [Zn(SCys)₄] site     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.