A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

Crystallization and preliminary diffraction data for iso-1-cytochrome c from yeast.

Deep red crystals of the electron transfer protein, iso-1-cytochrome c from yeast (Saccharomyces cerevisiae), have been obtained from a 90% saturated solution of (NH4)2SO4 containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2.0 A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained.

The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates.

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.

High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c.

The structure of yeast iso-1-cytochrome c has been refined against X-ray diffraction data to a nominal resolution of 1.23 A. The atomic model contains 893 protein atoms, as well as 116 water molecules and one sulfate anion. Also included in the refinement are 886 hydrogen atoms belonging to the protein molecule. The crystallographic R-factor is 0.192 for the 12,513 reflections with F greater than or equal to 3 sigma (F) in the resolution range 6.0 to 1.23 A. Co-ordinate accuracy is estimated to be better than 0.18 A. The iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into a series of alpha-helices and reverse turns that serve to envelop the heme prosthetic group in a hydrophobic pocket. Inspection of the conformations of helices in the molecule shows that the local environments of the helices, in particular the presence of intrahelical threonine residues, cause distortions from ideal alpha-helical geometry. Analysis of the internal mobility of iso-1-cytochrome c, based on refined crystallographic temperature factors, shows that the most rigid parts of the molecule are those that are closely associated with the heme group. The degree of saturation of hydrogen-bonding potential is high, with 90% of all polar atoms found to participate in hydrogen bonding. The geometry of intramolecular hydrogen bonds is typical of that observed in other high-resolution protein structures. The 116 water molecules present in the model represent about 41% of those expected to be present in the asymmetric unit. The majority of the water molecules are organized into a small number of hydrogen-bonding networks that are anchored to the protein surface. Comparison of the structure of yeast iso-1-cytochrome c with those of tuna and rice cytochromes c shows that these three molecules have very high structural similarity, with the atomic packing in the heme crevice region being particularly highly conserved. Large conformational differences that are observed between these cytochromes c can be explained by amino acid substitutions. Additional subtle differences in the positioning of the side-chains of several highly conserved residues are also observed and occur due to unique features in the local environments of each cytochrome c molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

High-resolution study of the three-dimensional structure of horse heart metmyoglobin.

The three-dimensional structure of horse heart metmyoglobin has been refined to a final R-factor of 15.5% for all observed data in the 6.0 to 1.9 A resolution range. The final model consists of 1242 non-hydrogen protein atoms, 154 water molecules and one sulfate ion. This structure has nearly ideal bonding and bond angle geometry. A Luzzati plot of the variation in R-factor with resolution yields an estimated mean co-ordinate error of 0.18 A. An extensive analysis of the pattern of hydrogen bonds formed in horse heart metmyoglobin has been completed. Over 80% of the polypeptide chain is involved in eight helical segments, of which seven are composed mainly of alpha-helical (3.6(13))-type hydrogen bonds; the remaining helix is composed entirely of 3(10) hydrogen bonds. Altogether, of 102 hydrogen bonds between main-chain atoms only six are not involved in helical structures, and four of these six occur within beta-turns. The majority of water molecules in horse heart metmyoglobin are found in solvent networks that range in size from two to 35 members. The size of water molecule networks can be rationalized on the basis of three factors: the number of hydrogen bonds to the protein surface, the presence of charged side-chain atoms, and the ability to bridge to neighboring molecules in the crystal lattice. Bridging water networks form the dominant intermolecular interactions. The backbone conformation of horse heart metmyoglobin is very similar to sperm whale metmyoglobin, with significant differences in secondary structure occurring only near residues 119 and 120, where residues 120 to 123 in sperm whale form a distorted type I reverse turn and the horse heart protein has a type II turn at residues 119 to 122. Nearly all of the hydrogen bonds between main-chain atoms (occurring mainly in helical regions) are common to both proteins, and more than half of the hydrogen bonds involving side-chain atoms observed in horse heart are also found in sperm whale metmyoglobin. Unlike sperm whale metmyoglobin, the heme iron atom in horse heart metmyoglobin is not significantly displaced from the plane of the heme group.

A polypeptide chain-refolding event occurs in the Gly82 variant of yeast iso-1-cytochrome c.

The replacement of Phe82 in yeast iso-1-cytochrome c by a glycine residue substantially alters both the tertiary structure and electron transfer properties of this protein. The largest structural change involves a polypeptide chain refolding of residues 79 through 85. Refolding places glycines 82, 83 and 84 immediately adjacent to the plane of the heme group in a spatial positioning comparable to that of the phenyl ring of Phe82 in the wild-type protein. Despite this perturbation in structure, solvent accessibility computations show that heme solvent exposure has not increased in the Gly82 variant protein. However, refolding does result in the introduction of a number of polar groups into the hydrophobic heme pocket. This appears to be responsible for the decreased reduction potential of the heme in this protein. The present study, along with that of the Ser82 variant protein (Louie et al., 1988b), clearly establishes the link between dielectric constant within the heme crevice and reduction potential. The further anomalously low electron transfer activity of the Gly82 variant protein would appear to arise from two factors. First, the polypeptide chain medium now adjacent to the heme is unable to facilitate electron transfer in a manner similar to that of the aromatic side-chain of Phe82. Second, polypeptide chain refolding significantly alters the surface contour of the Gly82 protein rendering it less suitable to interact with the corresponding complementary surfaces of redox partners. Our data support the conclusion that Phe82 plays a number of roles in the electron transfer process mediated by yeast iso-1-cytochrome c. These include the maintenance of the heme environment, provision of an optimal medium along the path of electron transfer and formation of interactions at the contact interface in complexes with redox partners.

Refined structure of the gene 5 DNA binding protein from bacteriophage fd.

The three-dimensional structure of the gene 5 DNA binding protein (G5BP) from bacteriophage fd has been determined from a combination of multiple isomorphous replacement techniques, partial refinements and deleted fragment difference Fourier syntheses. The structure was refined using restrained parameter least-squares and difference Fourier methods to a final residual of R = 0.217 for the 3528 statistically significant reflections present to 2.3 A resolution. In addition to the 682 atoms of the protein, 12 solvent molecules were included. We describe here the dispositions and orientations of the amino acid side-chains and their interactions as visualized in the G5BP structure. The G5BP monomer of 87 peptide units is almost entirely in the beta-conformation, organized as a three-stranded sheet, a two-stranded beta-ribbon and a broad connecting loop. There is no alpha-helix present in the molecule. Two G5BP monomers are tightly interlocked about an intermolecular dyad axis to form a compact dimer unit of about 55 A X 45 A X 36 A. The dimer is characterized by two symmetry-related antiparallel clefts that traverse the monomer surfaces essentially perpendicular to the dyad axis. From the three-stranded antiparallel beta-sheet, formed from the first two-thirds of the sequence, extend three tyrosine residues (26, 34, 41), a lysine (46) and two arginine residues (16, 21) that, as indicated by other physical and chemical experiments, are directly involved in DNA binding. Other residues likely to share binding responsibility are arginine 80 extending from the beta-ribbon and phenylalanine 73 from the tip of this loop, but as provided, however, by the opposite monomer within each G5BP dimer pair. Thus, both symmetry-related DNA binding sites have a composite nature and include contributions from both elements of the dimer. The gene 5 dimer is clearly the active binding species, and the two monomers within the dyad-related pair are so structurally contiguous that one cannot be certain whether the isolated monomer would maintain its observed crystal structure. This linkage is manifested primarily as a skeletal core of hydrophobic residues that extends from the center of each monomer continuously through an intermolecular beta-barrel that joins the pair. Protruding from the major area of density of each monomer is an elongated wing of tenuous structure comprising residues 15 through 32, which is, we believe, intimately involved in DNA binding. This wing appears to be dynamic and mobile, even in the crystal and, therefore, is likely to undergo conformational change in the presence of the ligand.

Oxidation state-dependent conformational changes in cytochrome c.

High-resolution three-dimensional structural analyses of yeast iso-1-cytochrome c have now been completed in both oxidation states using isomorphous crystalline material and similar structure determination methodologies. This approach has allowed a comprehensive comparison to be made between these structures and the elucidation of the subtle conformational changes occurring between oxidation states. The structure solution of reduced yeast iso-1-cytochrome c has been published and the determination of the oxidized protein and a comparison of these structures are reported herein. Our data show that oxidation state-dependent changes are expressed for the most part in terms of adjustments to heme structure, movement of internally bound water molecules and segmental thermal parameter changes along the polypeptide chain, rather than as explicit polypeptide chain positional shifts, which are found to be minimal. This result is emphasized by the retention of all main-chain to main-chain hydrogen bond interactions in both oxidation states. Observed thermal factor changes primarily affect four segments of polypeptide chain. Residues 37-39 show less mobility in the oxidized state, with Arg38 and its side-chain being most affected. In contrast, residues 47-59, 65-72 and 81-85 have significantly higher thermal factors, with maximal increases being observed for Asn52, Tyr67 and Phe82. The side-chains of two of these residues are hydrogen bonded to the internally bound water molecule, Wat166, which shows a large 1.7 A displacement towards the positively charged heme iron atom in the oxidized protein. Further analyses suggest that Wat166 is a major factor in stabilizing both oxidation states of the heme through differential orientation of dipole moment, shift in distance to the heme iron atom and alterations in the surrounding hydrogen bonding network. It also seems likely that Wat166 movement leads to the disruption of the hydrogen bond from the side-chain of Tyr67 to the Met80 heme ligand, thereby further stabilizing the positively charged heme iron atom in oxidized cytochrome c. In total, there appear to be three regions about which oxidation state-dependent structural changes are focussed. These include the pyrrole ring A propionate group, Wat166 and the Met80 heme ligand. All three of these foci are linked together by a network of intermediary interactions and are localized to the Met80 ligand side of the heme group. Associated with each is a corresponding nearby segment of polypeptide chain having a substantially higher mobility in the oxidized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure of RNase A complexed with d(pA)4.

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 A resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and preliminary diffraction data for horse heart metmyoglobin.

Reddish-brown crystals of metmyoglobin from horse heart have been obtained by both the hanging drop and batch crystallization methods in the space group P2(1), having a = 64.3 A, b = 28.9 A and c = 35.9 A, with beta = 107.1 degree. Morphologically similar crystal forms have been obtained for three derivatives of horse heart myoglobin having modified heme prosthetic groups.

Yeast iso-1-cytochrome c. A 2.8 A resolution three-dimensional structure determination.

A molecular replacement approach, augmented with the results of predictive modeling procedures, solvent accessibility studies, packing analyses and translational coefficient searches, has been used to elucidate the 2.8 A (1 A = 0.1 nm) resolution structure of yeast iso-1-cytochrome c. An examination of the polypeptide chain folding of this protein shows it to have unique conformations in three regions, upon comparison with the structures of other eukaryotic cytochromes c. These include: residues -5 to +1 at the N-terminal end of the polypeptide chain, which are in an extended conformation and project in large part off the surface of the protein; residues 19 to 26, which form a surface beta-loop on the His18 ligand side of the central heme group; and, the C-terminal end of the helical segment composed of residues 49 to 56, which serves to form a part of the heme pocket. Structural studies also show that the highly reactive sulfhydryl group of Cys102 is buried within a hydrophobic region in the monomer form of yeast iso-1-cytochrome c. Dimerization of yeast iso-1-cytochrome c through disulfide bond formation between two such residues would require a substantial conformational change in the C-terminal helix of this protein. Another unique structural feature, the trimethylated side-chain of Lys72, is located on the surface of yeast iso-1-cytochrome c near the solvent-exposed edge of the bound heme prosthetic group. On the basis of the results of these and other structural studies, an analysis of the spatial conservation of structural features in the heme pocket of eukaryotic cytochromes c has been conducted. It was found that the residues involved could be divided into three general classes. The current structural analyses and additional modeling studies have also been used to explain the altered functional properties observed for mutant yeast iso-1-cytochrome c proteins.

Mutation of tyrosine-67 to phenylalanine in cytochrome c significantly alters the local heme environment.

The high resolution three-dimensional atomic structures of the reduced and oxidized states of the Y67F variant of yeast iso-1-cytochrome c have been completed. The conformational differences observed are localized directly in the mutation site and in the region about the pyrrole A propionate. Shifts in atomic positions are largely restricted to nearby amino acid side-chains, whereas little perturbation of the polypeptide chain backbone is observed. One prominent difference between the variant and wild-type structures involves a substantial increase in the size of an already existing internal cavity adjacent to residue 67. This same cavity contains an internally bound water molecule (Wat166), which is found in all eukaryotic cytochromes c for which structures are available. In the reduced Y67F mutant protein a second water molecule (Wat300) is observed to reside in this enlarged internal cavity, assuming a position approximately equivalent to that of the hydroxyl group of Tyr67 in the wild-type protein. A further consequence of this mutation is the alteration of the hydrogen bond network between Tyr67, Wat166 and other nearby residues. This appears to be responsible for the absence of oxidation state dependent changes in polypeptide chain flexibility observed in the wild-type protein. Furthermore, loss of the normally resident Tyr67 OH to Met80 SD hydrogen bond leads to a significantly lower midpoint reduction potential. These results reaffirm proposals that both Tyr67 and Wat166 play a central role in stabilizing the alternative oxidation states of cytochrome c.

Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure.

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

High-resolution three-dimensional structure of horse heart cytochrome c.

The 1.94 A resolution three-dimensional structure of oxidized horse heart cytochrome c has been elucidated and refined to a final R-factor of 0.17. This has allowed for a detailed assessment of the structural features of this protein, including the presence of secondary structure, hydrogen-bonding patterns and heme geometry. A comprehensive analysis of the structural differences between horse heart cytochrome c and those other eukaryotic cytochromes c for which high-resolution structures are available (yeast iso-1, tuna, rice) has also been completed. Significant conformational differences between these proteins occur in three regions and primarily involve residues 22 to 27, 41 to 43 and 56 to 57. The first of these variable regions is part of a surface beta-loop, whilst the latter two are located together adjacent to the heme group. This study also demonstrates that, in horse cytochrome c, the side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a conformation comparable to that found in other cytochromes c. The positioning of this residue does not therefore appear to be oxidation-state-dependent. In total, five water molecules occupy conserved positions in the structures of horse heart, yeast iso-1, tuna and rice cytochromes c. Three of these are on the surface of the protein, serving to stabilize local polypeptide chain conformations. The remaining two are internally located. One of these mediates a charged interaction between the invariant residue Arg38 and a nearby heme propionate. The other is more centrally buried near the heme iron atom and is hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78. It is shown that this latter water molecule shifts in a consistent manner upon change in oxidation state if cytochrome c structures from various sources are compared. The conservation of this structural feature and its close proximity to the heme iron atom strongly implicate this internal water molecule as having a functional role in the mechanism of action of cytochrome c.

Crystallization of yeast iso-2-cytochrome c using a novel hair seeding technique.

A hair seeding technique has been developed to obtain diffraction quality crystals of yeast (Saccharomyces cerevisiae) iso-2-cytochrome c, a model for studies of protein folding and biological electron transfer reactions. Deep red crystals of this protein were obtained from 88 to 92% saturated solutions of ammonium sulfate containing 20 mg protein/ml, 0.1 M-sodium phoshate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to phosphate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to pH 6.0. Rapid crystal growth was observed, but only along the path of the seeding hair stroke. The space group is P4(3)2(1)2 (or P4(1)2(1)2) with a = b = 36.4 A, c = 137.8 A (1 A = 0.1 nm) and Z = 8. Crystals are stable in the X-ray beam for more than 10 days and diffract to at least 2.5 A resolution. The same hair seeding methodology has proven useful in obtaining crystals of specifically designed mutant iso-2 proteins and in other protein systems where consistent crystal growth had previously proven difficult to attain.

Structure determination and analysis of yeast iso-2-cytochrome c and a composite mutant protein.

As part of a study of protein folding and stability, the three-dimensional structures of yeast iso-2-cytochrome c and a composite protein (B-2036) composed of primary sequences of both iso-1 and iso-2-cytochromes c have been solved to 1.9 A and 1.95 A resolutions, respectively, using X-ray diffraction techniques. The sequences of iso-1 and iso-2-cytochrome c share approximately 84% identity and the B-2036 composite protein has residues 15 to 63 from iso-2-cytochrome c with the rest being derived form the iso-1 protein. Comparison of these structures reveals that amino acid substitutions result in alterations in the details of intramolecular interactions. Specifically, the substitution Leu98Met results in the filling of an internal cavity present in iso-1-cytochrome c. Further substitutions of Val20Ile and Cys102Ala alter the packing of secondary structure elements in the iso-2 protein. Blending the isozymic amino acid sequences in this latter area results in the expansion of the volume of an internal cavity in the B-2036 structure to relieve a steric clash between Ile20 and Cys102. Modification of hydrogen bonding and protein packing without disrupting the protein fold is illustrated by the His26Asn and Asn63Ser substitutions between iso-1 and iso-2-cytochromes c. Alternatively, a change in main-chain fold is observed at Gly37 apparently due to a remote amino acid substitution. Further structural changes occur at Phe82 and the amino terminus where a four residue extension is present in yeast iso-2-cytochrome c. An additional comparison with all other eukaryotic cytochrome c structures determined to date is presented, along with an analysis of conserved water molecules. Also determined are the midpoint reduction potentials of iso-2 and B-2036 cytochromes c using direct electrochemistry. The values obtained are 286 and 288 mV, respectively, indicating that the amino acid substitutions present have had only a small impact on the heme reduction potential in comparison to iso-1-cytochrome c, which has a reduction potential of 290 mV.

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase.

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.

The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c.

High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-1-cytochrome c variants have been completed in both oxidation states. The most prominent structural difference observed in both mutant proteins is the displacement of a conserved, internally bound water molecule (Wat166) from the protein matrix. In wild-type yeast iso-1-cytochrome c the position and orientation of this water molecule is found to be dependent on the oxidation state of the heme iron atom. Overall our results suggest the function of Wat166 and its associated hydrogen bond network is threefold. First, the presence of Wat166 provides a convenient mechanism to modify the hydrogen bond network involving several residues near the Met80 ligand in an oxidation state dependent manner. Second, Wat166 is necessary for the maintenance of the spatial relationships between nearby side-chains and the hydrogen bond interactions formed between these groups in this region of the protein. An essential part of this role is ensuring the proper conformation of the side-chain of Tyr67 so that it forms a hydrogen bond interaction with the heme ligand Met80. This hydrogen bond influences the electron withdrawing power of the Met80 ligand and is therefore a factor in controlling the midpoint reduction potential of cytochrome c. Elimination of this interaction in the N52I-Y67F mutant protein or elimination of Wat166 in the N52I protein with the subsequent disruption in the position and interactions of the Tyr67 side-chain, leads to a drop of approximately 56 mV in the observed midpoint reduction potential of the heme group. Third, Wat166 also appears to mediate increases in the mobility of three nearby segments of polypeptide chain when cytochrome c is in the oxidized state. Previous studies have proposed these changes may be related to oxidation state dependent interactions between cytochrome c and its redox partners. Coincident with the absence of Wat166, such mobility changes are not observed in the N52I and N52I-Y67F mutant proteins. It is possible that much of the increased protein stability observed for both mutant proteins may be due to this factor. Finally, our results show that neither heme iron charge nor heme plane distortion are responsible for oxidation state dependent conformational changes in the pyrrole A propionate region. Instead, the changes observed appear to be driven by the change in conformation that the side-chain of Asn52 experiences as the result of oxidation state dependent movement of Wat166.

Effect of the Asn52----Ile mutation on the redox potential of yeast cytochrome c. Theory and experiment.

Theoretical methods for correlation of sequence changes and redox potential of electron transport proteins are examined using the Asn52----Ile mutation in cytochrome c as a test case. The first approach uses the protein dipoles Langevin dipoles (PDLD) method and the high resolution X-ray structures of the native and the mutant proteins. This approach is found to give reliable results where all the solvent molecules are represented by Langevin dipoles and also when some bound water molecules are represented explicitly. A free energy perturbation method is also found to give reasonable results but at the expense of much more computer time. Finally, an approach that generates mutant structures from the native structure by molecular dynamics simulation and then uses these configurations in PDLD calculations is found to give a reasonable estimate of the effect of the mutation on the corresponding redox potential. The encouraging results obtained here and in a preliminary test case of the Phe82----Ser mutation indicates that the present strategies can provide a useful tool for structure-redox and sequence-redox correlation in proteins.