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G A Petsko

Publications and source records attributed to G A Petsko.

At least 163 records · Page 9Linked to original sources
PubMed1986

Study of protein dynamics by X-ray diffraction.

Properly carried out, high-resolution X-ray diffraction data collection followed by careful least-squares refinement can give the spatial distribution of the high-frequency mean-square displacements in a protein. These displacements reflect both individual atomic fluctuations in hard variables (bond lengths and bond angles) and collective motions involving soft variables (torsion angles, nonbonded interactions). Lower frequency, large amplitude motions and rapid but improbable motions are not quantifiable, but they may lead to such complete disorder that their existence can at least be inferred from the absence of interpretable electron density for some sections of the structure. Interior residues are more rigid than groups on the surface, and structural constraints are reflected in restricted motion even for surface residues. Amplitudes of motion of 0.5 A or greater are not uncommon. The temperature dependence of these fast motions varies considerably over the structure. In general, large [chi 2] values have large temperature dependence, while small displacements are less affected by temperature; however, exceptions are common. Significant reduction in [chi 2] on cooling establishes that proteins are mobile even in the crystalline state, and that static disorder is not the dominant contributor to the individual mean square displacements. Disordered regions in electron density maps are no longer automatically taken as signs of errors in structure determination. It is now recognized that the absence of strong electron density is often an indicator of conformational flexibility. Some of the functional roles for protein dynamics are beginning to be understood. Missing from these results are the physicochemical details that can be extracted from thermal motion analysis of small molecule crystal structures. Application of these methods to protein data is very difficult, but it is well to remember that just over 10 years ago it was commonly felt that protein structures could not even be refined. Certainly some small, well-diffracting proteins should be amenable to many of the sophisticated small-molecule analyses, as they yield X-ray data to resolutions comparable to simple organic structures. The most important type of analysis that awaits is anisotropic B factor refinement, which would give the principal directions of motion added to the amplitude information now obtained. Unfortunately, refinement of unrestrained anisotropic thermal elipsoids requires six parameters for each atom instead of a single isotropic B parameter, and even 1.5 A resolution data do not provide enough overdeterminacy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Aromatic-aromatic interaction: a mechanism of protein structure stabilization.

Analysis of neighboring aromatic groups in four biphenyl peptides or peptide analogs and 34 proteins reveals a specific aromatic-aromatic interaction. Aromatic pairs (less than 7 A between phenyl ring centroids) were analyzed for the frequency of pair type, their interaction geometry (separation and dihedral angle), their nonbonded interaction energy, the secondary structural locations of interacting residues, their environment, and their conservation in related molecules. The results indicate that on average about 60 percent of aromatic side chains in proteins are involved in aromatic pairs, 80 percent of which form networks of three or more interacting aromatic side chains. Phenyl ring centroids are separated by a preferential distance of between 4.5 and 7 A, and dihedral angles approaching 90 degrees are most common. Nonbonded potential energy calculations indicate that a typical aromatic-aromatic interaction has energy of between -1 and -2 kilocalories per mole. The free energy contribution of the interaction depends on the environment of the aromatic pair. Buried or partially buried pairs constitute 80 percent of the surveyed sample and contribute a free energy of between -0.6 and -1.3 kilocalories per mole to the stability of the protein's structure at physiologic temperature. Of the proteins surveyed, 80 percent of these energetically favorable interactions stabilize tertiary structure, and 20 percent stabilize quaternary structure. Conservation of the interaction in related molecules is particularly striking.

Amino Acids↗
PubMed1984

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase.

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.

Animals↗
PubMed1984

Cavities in proteins: structure of a metmyoglobin-xenon complex solved to 1.9 A.

X-ray crystallographic data to 1.9-A resolution were collected on sperm whale metmyoglobin equilibrated with 7 atm of xenon gas. The results indicate four xenon sites of occupancy from 0.45 to 1.0. These sites are located in interior spaces or packing defects of the myoglobin molecule. The effects of the bound xenon on the protein structure are minor, and we observe a small overall reduction in refined isotropic atomic protein temperature factors. We interpret the results as a confirmation that, on a time-averaged basis, cavities exist within the myoglobin molecule and suggest that the binding of small ligands in these cavities affects the internal motions and conformational substrates of the protein.

Animals↗
PubMed1984

Reaction of myoglobin with phenylhydrazine: a molecular doorstop.

X-ray crystallographic studies of myoglobin do not show an entrance or exit path for potential ligands from the surface to the heme cavity. Efforts to locate such a path have so far centered around dynamic calculations. A structure has now been determined that has a clear opening. Phenylhydrazine reacts with myoglobin in such a way that a phenyl group remains bound to the iron atom. The structure of this complex shows that the side chains of His-64(E7), Arg-45-(CD3), and Val-68(E11) have been forced aside to form an open channel to the surface. Although this may not be the only channel to the iron atom, it seems likely that it is an important one.

Heme↗
PubMed1983

The x-ray structure of the periplasmic galactose binding protein from Salmonella typhimurium at 3.0-A resolution.

The x-ray structure of the periplasmic galactose binding protein from Salmonella typhimurium, the specific receptor for taxis toward, and high-affinity transport of, galactose has been solved at 3.0-A resolution using multiple isomorphous replacement. The path of the polypeptide chain has been traced, and a model structure consisting of 292 amino acids has been fit to the electron density map. The overall shape of the molecule is that of a prolate ellipsoid, with dimensions 35 X 35 X 65 A. The protein consists of two similar domains of roughly equal size, related by an axis of pseudosymmetry, and separated by a deep cleft about 8 A wide. Each domain has a core of parallel beta sheet surrounded by five alpha helices, built by alternating strands of sheet and helix in a repeating pattern. Approximately 36% of the residues are involved in alpha helices, and 27% in beta sheet. The tertiary structure has been compared to that of the Escherichia coli arabinose binding protein (Gilliland, G.L., and Quiocho, F. A. (1981) J. Mol. Biol. 146, 341-362), a periplasmic receptor which is involved in transport, but not in chemotaxis. The overall folding of these two molecules is very similar, with the exception of two areas on the surface of the molecule on the long sides of the prolate ellipsoid. The observed variations are adequate to explain the differences in interaction of L-arabinose binding protein and galactose binding protein with the membrane proteins for transport and chemotaxis.

Calcium-Binding Proteins↗
PubMed1983

The introduction of specific sites for heavy metal binding in a crystalline protein.

Heavy metal derivatives of the galactose binding protein of Salmonella typhimurium were obtained by the treatment of crystals with carbon disulfide under anaerobic conditions, followed by exposure to mercury-containing reagents. Carbon disulfide reacts with protein amino groups to give a metastable dithiocarbamate, which is susceptible to covalent derivatization by mercurials. The number of amino groups which react for any particular crystalline protein will depend on the pH, the composition of the crystal mother liquor, and the steric accessibility limitations imposed by crystal packing. Direct reaction with protein crystals, rather than solution derivatization followed by purification and subsequent crystallization, is used to promote isomorphism of the derivative crystal with the native and to limit the number of available sites. For the S. typhimurium galactose binding protein, carbon disulfide treatment, followed by reaction with 2-chloromercuri-4-nitrophenol, resulted in binding at two sites at pH 8.0. Similar treatment with dimercury acetate gave one binding site for the dimercurial at the same pH. Both derivatives were isomorphous with the native crystal to a resolution of at least 3.5 A. These heavy atom derivatives have been used to produce an interpretable electron density map of the protein at 3-A resolution.

Calcium-Binding Proteins↗
PubMed1983

Structure of oxidized flavodoxin from Anacystis nidulans.

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

Amino Acid Sequence↗
PubMed1983

The iron content of iron superoxide dismutase: determination by anomalous scattering.

The number of iron atoms in the dimeric iron-containing superoxide dismutase from Pseudomonas ovalis and their atomic positions have been determined directly from anomalous scattering measurements on crystals of the native enzyme. To resolve the long-standing question of the total amount of iron per molecule for this class of dismutase, the occupancy of each site was refined against the measured Bijvoet differences. The enzyme is a symmetrical dimer with one iron site in each subunit. The iron position is 9 A from the intersubunit interface. The total iron content of the dimer is 1.2 +/- 0.2 moles per mole of protein. This is divided between the subunits in the ratio 0.65:0.55; the difference between them is probably not significant. Since each subunit contains, on average, slightly more than half an iron atom we conclude that the normal state of this enzyme is two iron atoms per dimer but that some of the metal is lost during purification of the protein. Although the crystals are obviously a mixture of holo- and apo-enzymes, the 2.9 A electron density map is uniformly clean, even at the iron site. We conclude that the three-dimensional structures of the iron-bound enzyme and the apo-enzyme are identical.

Apoenzymes↗
PubMed1983

Studies of calmodulin structure: laser raman spectroscopy of biomolecules.

The structure of bovine brain calmodulin was probed by using laser Raman spectroscopy to elucidate cation-induced conformational changes in the protein. Local changes, most likely reflecting metal binding but not rearrangement of the peptide backbone, were observed in the presence of calcium or magnesium. A conformational change involving the peptide backbone and secondary structure content of calmodulin was observed only in the presence of calcium. The calcium-induced conformational change in the peptide backbone involves increased alpha helix and beta sheet. This was the only major calcium-specific change observed in the Raman spectrum, which suggests that the flexibility of the backbone conformation may play a critical role in the physiological activity of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

The role of mobility in the substrate binding and catalytic machinery of enzymes.

Recent theoretical and experimental studies have demonstrated that proteins are fluctuating systems capable of large, seemingly random, excursions from the equilibrium conformation. Attention is now focusing on the functional consequences of these motions. X-ray diffraction is a powerful tool for mapping the spatial distribution of protein dynamics; studies on the temperature dependence of the apparent Debye-Waller factors of crystalline myoglobin demonstrate that proteins are flexible in the solid state. Crystallographic studies of a Michaelis complex of ribonuclease A show that a mobile lysine adapts its conformation to the changes in stereochemistry and charge distribution in the substrate during catalysis. The structure of the triose phosphate isomerase-substrate complex shows that a mobile region of 10 amino acids becomes ordered when ligand binds. These studies suggest several roles for protein mobility in enzymic catalysis: providing access to internal sites, allowing changes in substrate structure during the reaction, and reducing the observed binding constant of substrate and product to the enzyme by decreasing entropy. A flexible enzyme also does not need a communication system to signal binding or transformation, since a pre-existing equilibrium can be used. More speculative ideas, such as the guiding of thermal vibrations along the reaction coordinate, can only be tested when more detailed data are available.

Binding Sites↗