Biomedical subjects

I Rayment

Publications and source records attributed to I Rayment.

At least 73 records · Page 4Linked to original sources

PubMed1995

Structural studies of myosin:nucleotide complexes: a revised model for the molecular basis of muscle contraction.

The structures of the MgADP-beryllium fluoride and MgADP-aluminum fluoride complexes of the truncated myosin head from Dictyostelium myosin II are reported. These reveal the location of the nucleotide complex and define the amino acid residues that form the active site. The tertiary structure of the beryllium fluoride complex is essentially identical to that seen previously in the three-dimensional structure of chicken skeletal muscle myosin. By contrast, significant domain movements are observed in the aluminum fluoride complex. These structural findings form the basis of a revised model for the structural basis of the contractile cycle. It is now suggested that the narrow cleft that splits the central 50-kDa segment of the heavy chain provides not only the communication route between the nucleotide-binding pocket and actin but also transmits the conformational change necessary for movement.

Adenosine Diphosphate↗

PubMed1995

Is myosin a "back door" enzyme?

ATP has been modeled into the active site of chicken skeletal myosin subfragment-1 using the adenylate kinase.Ap5A structure as a starting reference. The resulting docked ATP.S1 structure is justified in that it rationalizes the photolabeling data from several ATP analogs. The gamma-phosphate of ATP sits at the bottom of the active site pocket and is partially visible via a view along the prominent 50-kDa cleft of S1 but not when viewed from above the active site. It is postulated that actin binding promotes the movement of the P-loop and Arg-245 to allow Pi from ATP to leave via a "back-door" in the 50-kDa fragment while ADP is still bound at the active site. Such a mechanism can explain a number of experimental observations, including the kinetics of ATP hydrolysis, the nucleotide dependence of Pi exchange into ATP, and the formation of stable myosin.ADP.vanadate complexes in muscle fibers.

Adenosine Triphosphate↗

PubMed1994

Three-dimensional structure of the muscle fatty-acid-binding protein isolated from the desert locust Schistocerca gregaria.

The three-dimensional structure of the fatty-acid-binding protein isolated from the flight muscle of the desert locust Schistocerca gregaria has been solved and refined to a crystallographic R-value of 18.5% for all measured X-ray data from 30.0- to 2.2-A resolution. Crystals employed in the investigation were grown from 2.6 to 2.8 M ammonium sulfate solutions, buffered at pH 7.5 and containing 2-5% 2-methyl-2,4-pentanediol. They belonged to the space group P2(1) with unit cell dimensions of a = 61.6 A, b = 44.8 A, c = 63.9 A, and beta = 113.6 degrees and two molecules per asymmetric unit. The protein fold consists of ten strands of antiparallel beta-pleated sheet that wrap around to form a beta-barrel. In addition, there are two small alpha-helices and six type I, two type II, and two type II' turns. The two molecules pack in the asymmetric unit as a dimer with a local 2-fold rotational axis. The subunit-subunit interface involves amino acid side chains located in the area of the helix-turn-helix motif and the turn between beta-strands E and F. It is this area that has been speculated to form the portal through which fatty acids enter the binding cavity. There are 23 solvent molecules that are conserved between the two independent molecules in the asymmetric unit. Nine of these waters play important structural roles. A three-dimensional comparison between the insect and human muscle fatty-acid-binding proteins shows that their alpha-carbons superimpose with a root-mean-square deviation of 0.77 A for 89 structurally equivalent atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase is found in all animals, plants, and bacteria and catalyzes the first committed step in fatty acid synthesis. It is a multicomponent enzyme containing a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase functionality. Here we report the X-ray structure of the biotin carboxylase component from Escherichia coli determined to 2.4-A resolution. The structure was solved by a combination of multiple isomorphous replacement and electron density modification procedures. The overall fold of the molecule may be described in terms of three structural domains. The N-terminal region, formed by Met 1-Ile 103, adopts a dinucleotide binding motif with five strands of parallel beta-sheet flanked on either side by alpha-helices. The "B-domain" extends from the main body of the subunit where it folds into two alpha-helical regions and three strands of beta-sheet. Following the excursion into the B-domain, the polypeptide chain folds back into the body of the protein where it forms an eight-stranded antiparallel beta-sheet. In addition to this major secondary structural element, the C-terminal domain also contains a smaller three-stranded antiparallel beta-sheet and seven alpha-helices. The active site of the enzyme has been identified tentatively by a difference Fourier map calculated between X-ray data from the native crystals and from crystals soaked in a Ag+/biotin complex. Those amino acid residues believed to form part of the active site pocket include His 209-Glu 211, His 236-Glu 241, Glu 276, Ile 287-Glu 296, and Arg 338.2+ represents the first X-ray model of a biotin-dependent carboxylase.

Acetyl-CoA Carboxylase↗

PubMed1994

Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A resolution.

The structure of a new crystal form of enolase from bakers' yeast has been solved to 2.1-A resolution. Crystals were grown from poly(ethylene glycol) and KCl at pH 8.2 in the presence of Mg2+ and a reaction intermediate analog, phosphonoacetohydroxamate (PhAH). Crystals belong to space group C2; have unit cell dimensions a = 123.5 A, b = 73.9 A, and c = 94.8 A with beta = 93.3 degrees; and contain one dimer per asymmetric unit. The structure was solved by molecular replacement from the X-ray coordinates of apoenolase [Stec, B., & Lebioda, L. (1990) J. Mol. Biol. 211, 235-248]. Both essential divalent metal ions are observed to be complexed with the inhibitor. The two Mg2+ ions are 4.05 A apart and are bridged by a mu-oxyl ligand from the carbonyl moiety of PhAH. The "high-affinity" Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, one water molecule, and the hydroxamate and carbonyl oxygens of PhAH. The second Mg2+ coordinates to a phosphonyl oxygen, two water molecules, and the mu-bridge carbonyl oxygen of PhAH. Coordination schemes with respect to PhAH and water ligands are fully consistent with those of the Mn2+ complexes determined spectroscopically [Poyner, R.R., & Reed, G. H. (1992) Biochemistry 31, 7166-7173]. Remaining ligands for the second Mg2+ are the carbonyl oxygen and gamma-oxygen of Ser 39. Chelation of this Ser residue to Mg2+ effectively "latches" a flexible loop extending from Gly 37 through His 43 and closes off the entrance to the active site. The position of the second Mg2+ in the active site provides new insight into the stereochemistry of substrate binding.

Amino Acid Sequence↗

PubMed1994

Structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate.

The molecular structure of rabbit muscle pyruvate kinase, crystallized as a complex with Mn2+, K+, and pyruvate, has been solved to 2.9-A resolution. Crystals employed in the investigation belonged to the space group P1 and had unit cell dimensions a = 83.6 A, b = 109.9 A, c = 146.8 A, alpha = 94.9 degrees, beta = 93.6 degrees, and gamma = 112.3 degrees. There were two tetramers in the asymmetric unit. The structure was solved by molecular replacement, using as the search model the coordinates of the tetramer of pyruvate kinase from cat muscle [Muirhead, H., Claydon, D. A., Barford, D., Lorimer, C. G., Fothergill-Gilmore, L. A., Schiltz, E., & Schmitt, W. (1986) EMBO J.5, 475-481]. The amino acid sequence derived from the cDNA coding for the enzyme from rabbit muscle was fit to the electron density. The rabbit and cat muscle enzymes have approximately 94% sequence identity, and the folding patterns are expected to be nearly identical. There are, however, three regions where the topological models of the cat and rabbit pyruvate kinases differ. Mn2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. These two residues are strictly conserved in all known pyruvate kinases. In addition, the density for Mn2+ is connected to that of pyruvate, consistent with chelation through a carboxylate oxygen and the carbonyl oxygen of the substrate. The epsilon-NH2 of Lys 269 and the OH of Thr 327 lie on either side of the methyl group of bound pyruvate. Spherical electron density, assigned to K+, is located within a well-defined pocket of four oxygen ligands contributed by the carbonyl oxygen of Thr 113, O gamma of Ser 76, O delta 1 of Asn 74, and O delta 2 of Asp 112. The interaction of Asp 112 with the side chains of Lys 269 and Arg 72 may mediate, indirectly, monovalent cation effects on activity.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary crystallographic analysis of galactose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate uridylyltransferase catalyzes the formation of UDP-galactose during normal cellular metabolism, making it an essential enzyme in all cells. The enzyme from Escherichia coli has been crystallized at pH 5.9 in the presence of phenyl-UDP (P(1)-5'-uridyl-P(2)-phenyl diphosphate), a substrate analog, using PEG 10 000 in combination with Li(2)SO(4) and NaCl. Crystals belong to space group P2(1)2(1)2 with unit-cell dimensions a = 58.6, b = 217.6 and c = 69.6 A. There is one dimer or two subunits in the asymmetric unit. Crystals are relatively insensitive to X-ray radiation and diffract beyond 2.5 A resolution. A low-resolution native data set has been recorded.

Journal Article↗

PubMed1994

Molecular structure of the oxidized high-potential iron-sulfur protein isolated from Ectothiorhodospira vacuolata.

The high-potential iron-sulfur protein (iso-form II) isolated from Ectothiorhodospira vacuolata has been crystallized and its three-dimensional structure determined by molecular replacement procedures and refined to 1.8-A resolution with a crystallographic R factor of 16.3%. Crystals employed in the investigation belonged to the space group C222(1) with unit cell dimensions of a = 58.4 A, b = 64.7 A, and c = 39.3 A and one molecule per asymmetric unit. Like those HiPIPs structurally characterized thus far, the E. vacuolata molecule contains mostly reverse turns that wrap around the iron-sulfur cluster with cysteine residues 34, 37, 51, and 65 ligating the metal center to the polypeptide chain. There are 57 ordered solvent molecules, most of which lie at the surface of the protein. Two of these water molecules play important structural roles by stabilizing the loops located between Asp 42 and Lys 57. The metal center binding pocket is decidedly hydrophobic with the closest solvent molecule being 6.9 A from S2 of the [4Fe-4S] cluster. The E. vacuolata HiPIP molecules pack in the crystalline lattice as dimers with their iron-sulfur centers approximately 17.5 A apart. On the basis of biochemical properties, it was anticipated that the E. vacuolata HiPIP would be structurally more similar to the HiPIP isolated from Ectothiorhodospira halophila than to the protein obtained from Chromatium vinosum. In fact, the E. vacuolata molecule is as structurally close to the C. vinosum HiPIP as it is to the E. halophila protein due to the presence of various insertions and deletions that disrupt local folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Preliminary X-ray crystallographic analysis of biotin carboxylase isolated from Escherichia coli.

Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids. In Escherichia coli, the enzyme consists of three subunits that are isolated separately and display distinct functional properties. Here we report the crystallization and preliminary X-ray analysis of one of these components, namely biotin carboxylase. The crystals are grown by microdialysis against 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 2 mM DTT and 1 mM NaN3 at 4 degrees C. They belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 61.9 A, b = 96.1 A and c = 180.6 A and contain one dimer per asymmetric unit. The crystals diffract to a nominal resolution of 2.2 A. From a mechanistic standpoint, biotin carboxylase is especially interesting in that it is the smallest protein within its class and is one of only two carboxylases that can utilize free biotin as a substrate.

Acetyl-CoA Carboxylase↗

PubMed1994

The three-dimensional structure of a molecular motor.

Myosin is one of only three proteins known to convert chemical energy into mechanical work. Although the chemical, kinetic and physiological characteristics of this protein have been studied extensively, it has been difficult to define its molecular basis of movement. With the recent X-ray structural determination of the myosin head, however, it is now possible to put forward a hypothesis on how myosin might function as a molecular motor.

Actomyosin↗

PubMed1993

Molecular structure of kanamycin nucleotidyltransferase determined to 3.0-A resolution.

Kanamycin nucleotidyltransferase, as originally isolated from Staphylococcus aureus, inactivates the antibiotic kanamycin by catalyzing the transfer of a nucleotidyl group from nucleoside triphosphates such as ATP to the 4'-hydroxyl group of the aminoglycoside. The molecular structure of the enzyme described here was determined by X-ray crystallographic analysis to a resolution of 3.0 A. Crystals employed in the investigation belonged to the space group P4(3)2(1)2 with unit cell dimensions of a = b = 78.9 A and c = 219.2 A. An electron density map phased with seven heavy-atom derivatives revealed that the molecules packed in the crystalline lattice as dimers exhibiting local 2-fold rotation axes. Subsequent symmetry averaging and solvent flattening improved the quality of the electron density such that it was possible to completely trace the 253 amino acid polypeptide chain. Each monomer is divided into two distinct structural domains: the N-terminal motif composed of residues Met 1-Glu 127 and the C-terminal half delineated by residues Ala 128-Phe 253. The N-terminal region is characterized by a five-stranded mixed beta-pleated sheet whereas the C-terminal domain contains five alpha-helices, four of which form an up-and-down alpha-helical bundle very similar to that observed in cytochrome c'. The two subunits wrap about one another to form an ellipsoid with a pronounced cleft that could easily accommodate the various aminoglycosides known to bind to the enzyme.

Amino Acid Sequence↗

PubMed1993

Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution.

Chemical modification of proteins has been and continues to be an important biochemical tool for the study of protein structure and function. One such type of approach has been the reductive methylation of lysine residues. In order to address the consequences of such methylation on the crystallization and structural properties of a protein, the three-dimensional structure of hen egg white lysozyme in which all lysine residues have been alkylated has been determined and refined to a nominal resolution of 1.8 A and a crystallographic R factor of 17.3%. Crystals used in the investigation were grown from 1.5-1.8 M MgSO4 and 50 mM Tris at pH 8.0 and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 30.6 A, b = 56.3 A, c = 73.2 A, and one molecule per asymmetric unit. It was not possible to grow crystals of the modified lysozyme under the conditions normally employed for the hen egg white protein. Overall, the three-dimensional structures of the native lysozyme and the modified protein are very similar with only two surface loops differing to any significant extent. Specifically, the positions of the alpha-carbons for these two forms of the protein, excluding the surface loops, superimpose with a root-mean-square value of 0.40 A. The magnitude of the structural changes observed between the modified an unmodified forms of lysozyme is similar to that seen when an identical protein structure is solved in two different crystalline lattices.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Kinetic characterization of reductively methylated myosin subfragment 1.

Reductive methylation of myosin-S1 converts 97% of lysine residues in native myosin-S1 to dimethyllysine without detectable modification of other amino acid side chains. RM-S1 is catalytically active, although the rate and equilibrium constants of many of the steps of the actomyosin ATP hydrolysis mechanism have been altered. The steady-state rate of MgATP hydrolysis by modified myosin-S1 (RM-S1) is increased 4-5-fold in the absence of actin. However, the maximum steady-state rate of RM-S1 at saturating actin, 0.59 s-1, is less than one-tenth that observed for the unmodified protein, 7.4 s-1 (5 mM MOPS, 2 mM MgCl2, pH 7, 20 degrees C). Under single-turnover conditions [S1] > [ATP], the observed rate of ATP hydrolysis by RM-S1 is fit by a single exponential that is no more than twice the steady-state rate, which indicates that the bond splitting state is at least partially rate limiting for RM-S1. Although a small decrease in intrinsic tryptophan fluorescence is observed upon ATP binding to RM-acto-S1, the large and rapid fluorescence enhancement produced by aza-ATP binding to RM-acto-S1 is nearly normal. ATP binds and dissociates modified RM-S1 from actin with a second-order rate constant that is more than twice that observed for control S1. The changes in the kinetic mechanism produced by reductive methylation of lysine are qualitatively and quantitatively similar to the changes that are induced by either SH1 modification or substrate analogues such as GTP.

Actomyosin↗

PubMed1993

Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1.

Elucidation of the molecular contacts between actin and myosin is central to understanding the force-generating process in muscle and other cells. Actin, a highly conserved globular protein found in all eukaryotes, polymerizes into filaments (F-actin) for most of its biological functions. Myosins, which are more diverse in sequence, share a conserved globular head of about 900 amino acids in length (subfragment-1 or S1) at the N-terminal end of the molecule. S1 contains all the elements necessary for mechano-chemical force transduction in vitro. Here we report an atomic model for the actomyosin complex produced by combining the atomic X-ray structure of F-actin and chicken myosin S1 with a three-dimensional reconstruction from electron micrographs of frozen-hydrated F-actin decorated with recombinant Dictyostelium myosin S1. The accuracy of the reconstruction shows the position of actin and myosin molecules unambiguously.

Actins↗

PubMed1993

Molecular structure of the oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120 determined to 1.7-A resolution.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. The three-dimensional structure of this ferredoxin has now been determined and refined to a crystallographic R value of 16.7%, with all measured X-ray data from 30.0 to 1.7 A. The molecular motif of this ferredoxin is similar to that of other plant-type ferredoxins with the iron-sulfur cluster located toward the outer edge of the molecule and the irons tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteinyl residues. The overall secondary structure of the molecule consists of seven strands of beta-pleated sheet, two alpha-helices, and seven type I turns. It is of special interest that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different in the heterocyst form of the protein. Three of these positions are located in the metal-cluster binding loop.

Amino Acid Sequence↗

PubMed1993

Three-dimensional structure of myosin subfragment-1: a molecular motor.

Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

Actins↗

PubMed1993

Structure of the actin-myosin complex and its implications for muscle contraction.

Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP). A model for the rigor complex of F actin and the myosin head was obtained by combining the molecular structures of the individual proteins with the low-resolution electron density maps of the complex derived by cryo-electron microscopy and image analysis. The spatial relation between the ATP binding pocket on myosin and the major contact area on actin suggests a working hypothesis for the crossbridge cycle that is consistent with previous independent structural and biochemical studies.

Actins↗

PubMed1992

Three-dimensional structure of the high-potential iron-sulfur protein isolated from the purple phototrophic bacterium Rhodocyclus tenuis determined and refined at 1.5 A resolution.

The molecular structure of the high-potential iron-sulfur protein (HiPIP) isolated from the phototrophic bacterium, Rhodocyclus tenuis, has been solved and refined to a nominal resolution of 1.5 A with a crystallographic R-factor of 17.3% for all measured X-ray data from 30 A to 1.5 A. It is the smallest of the HiPIP structures studied thus far with 62 amino acid residues. Crystals used in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A and beta = 90.8 degrees and contained two molecules per asymmetric unit. The structure was solved by a combination of multiple isomorphous replacement with two heavy-atom derivatives, anomalous scattering from the iron-sulfur cluster, symmetry averaging and solvent flattening. The folding motif for this HiPIP is characterized by one small alpha-helix, six Type I turns, an approximate Type II turn and one Type I' turn. As in other HiPIPs, the iron-sulfur cluster is co-ordinated by four cysteinyl ligands and exhibits a cubane-like motif. These cysteinyl ligands are all located in Type I turns. The hydrogen bonding around the metal cluster in the R. tenuis protein is similar to the patterns observed in the Chromatium vinosum and Ectothiorhodospira halophila HiPIPs. Several of the amino acid residues invariant in the previously determined C. vinosum and E. halophila structures are not retained in the R. tenuis molecule. There are 13 solvent molecules structurally conserved between the two R. tenuis HiPIP molecules in the asymmetric unit, some of which are important for stabilizing surface loops. Interestingly, while it is assumed that this HiPIP functions as a monomer in solution, the two molecules in the asymmetric unit pack as a dimer and are related to each other by an approximate twofold rotation axis.

Bacterial Proteins↗