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F A Quiocho

Publications and source records attributed to F A Quiocho.

At least 145 records · Page 8Linked to original sources
PubMed1976

Structure of L-arabinose-binding protein from Escherichia coli at 5 A resolution and preliminary results at 3.5 A.

The three-dimensional crystal structure of the L-arabinose-binding protein from E. coli, an essential component in the active transport of L-arabinose, has been solved at 5 A resolution using the method of multiple isomorphous replacement. Five heavy atom derivatives were used. A preliminary 3.5 A electron density map has also been calculated. The results indicate that the molecule is ellipsoidal with approximate dimensions 68 A X 38 A X 30 A. Two similar domains within the molecule (which is a single polypeptide chain) are related by an approximate noncrystallographic rotation-translation axis. This relationship involves approximately 20% of the structure.

Arabinose↗
PubMed1972

Similarities between the conformation of arsanilazotyrosine 248 of carboxypeptidase A in the crystalline state and in solution.

Modification of carboxypeptidase A(gamma) crystals (Anson) with diazotized arsanilic acid specifically labels tyrosine 248; at pH 8.2 the modified enzyme gives yellow crystals, but a red solution. It has been suggested that arsanilazotyrosine 248 forms a complex with the Zn cofactor accounting for the red color in solution, but that a complex is not formed in the crystal. However, the crystal structure of carboxypeptidase A(gamma) is unknown. We show here that crystals of carboxypeptidase A(alpha), whose crystal structure has been determined, are red both in solution and in the crystalline state (at pH 8.2) after modification with diazotized arsanilic acid. These new data are of importance in relating the structure in the crystalline state to the catalytic mechanisms, as based on the x-ray diffraction evidence. The activity of carboxypeptidase A in the crystal and in solution has a ratio of only 1/3 for the alpha form, in contrast to the ratio of 1/300 for the gamma form, with carbobenzoxyglycyl-L-phenylalanine as a substrate.A pH-jump experiment monitored by stopped-flow kinetics in a split-beam apparatus has revealed a single exponential rate when a solution of arsanilazotyrosine 248 carboxypeptidase A(alpha) at pH 6.7 (yellow) is increased to pH 8.5 (red). The rate constants obtained in this experiment are 6.1 sec(-1) at 3.0 mg/ml and 7.2 sec(-1) at 1.6 mg/ml concentration of enzyme.

Animals↗
PubMed1971

Structure of soncanavalin A at 4 A resolution.

Concanavalin A, a phytohemagglutinin isolated from the jack bean, crystallizes at pH 6.8 in the orthorhombic space group 1222 with a = 89.9, b = 87.2, and c = 63.1 A. We have analyzed x-ray diffraction intensity data to 4 A resolution on native concanavalin A and five heavy-metal derivatives: lead, mersalyl, chloroplatinate, uranyl, and o-mercuri-p-nitrophenol. Heavy-atom positions, occupancies, and isotropic thermal parameters have been refined by least-squares methods. The electron density maps clearly show the molecular shape and the packing of the concanavalin A molecules. The asymmetric unit (mol wt 27,000) forms an elliptical dome or "gumdrop" with a base of approximately 46 x 26 A and a height of 42 A. The subunits are paired across 2-fold axes parallel to the c-axis to form dimers. The dimers are in turn paired across points of D(2) symmetry to form tetramers of roughly tetrahedral shape. Each unit has a depression located on the surface which could be the site of saccharide binding. In many regions we have been able to trace the course of the polypeptide chain.

Concanavalin A↗
PubMed1969

The structure of carboxypeptidase A. IX. The x-ray diffraction results in the light of the chemical sequence.

Several features of carboxypeptidase A (CPA) which were previously established by the X-ray diffraction structure studies have now been confirmed by the chemical sequence analysis. These results include the number (307) of amino acid residues in CPA(alpha), the identities of the residues (Arg 145, Glu 270, and Tyr 248) shown by the X-ray study to be involved in substrate binding and catalysis, and the existence of a disulfide bond. The Zn ligands, shown by the X-ray study to be residues 69, 72, and 196 and identified as His, Glx, and either Glx or Lys, are proved by the chemical sequence to be His, Glu, and His, respectively. No change is required in our previous mechanistic deductions, which are here extended to include a specific mechanism of activation of the substrate by a net charge on the metal ion, which suffers a change in local dielectric constant when it is covered by a substrate.

Amino Acid Sequence↗
PubMedDate not supplied

Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli.

The periplasmic maltose binding protein (MBP) is required for the high affinity transport of maltose and maltodextrins and for chemotaxis towards these sugars. In these functions, MBP interacts with proteins of the cytoplasmic membrane: MalF and MalG for transport, Tar for chemotaxis. A large number of MBP mutations have been isolated by us and other laboratories. We grouped these mutations into classes depending on the interactions affected and we represented the corresponding residues on the 3-D model for MBP so as to further identify the sites of MBP interacting with the MalF-MalG complex and with the Tar protein. MBP (like the other binding proteins) is composed of 2 lobes enclosing a cleft where the substrate binds. The face of the protein opposite the cleft seems to interact neither with MalF-MalG nor with Tar. The other face, corresponding to the cleft, contains sites for interactions with MalF-MalG and Tar. These sites appear to cover both sides of the cleft and may overlap in part. The present definition of the interaction sites suggests further that MBP has different in vivo orientations when it interacts with MalF-MalG or with Tar. This work constitutes an additional step in combining the use of genetic and structural analysis to define the interaction sites on MBP. Because of the structural similarities between periplasmic binding proteins, the regions of interaction defined could be relevant for other members of this family.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Novel stereospecificity of the L-arabinose-binding protein.

Tertiary structure refinement at 1.7 A resolution of the liganded form of L-arabinose-binding protein from Escherichia coli has revealed a novel binding site geometry which accommodates both alpha- and beta-anomers of L-arabinose. This detailed structure analysis provides new understanding of protein-sugar interaction, the process by which the binding protein minimizes the difference in the stability of the two bound sugar anomers, and the roles of periplasmic binding proteins in active transport.

Arabinose↗
PubMedDate not supplied

Predicted structure of the sugar-binding site of the lac repressor.

The lactose repressor protein from Escherichia coli binds sugars, primarily galactosides, which modulate its interactions with operator DNA and thereby affect synthesis of the lac metabolic enzymes. The affinity of the repressor for operator DNA is decreased by binding inducer sugars and increased by binding anti-inducer sugars. Based on regions of the primary structure implicated by genetic methods to be involved in sugar binding, amino acid sequence homology between L-arabinose-binding protein (ABP) and lac repressor has recently been reported. The sugar-binding sites for these two proteins might be expected to have similar structural features, as both bind L-arabinose and D-galactose. The high resolution structure of ABP reported in the accompanying article provides complete definition of amino acids in the sugar-binding site. By identification of homologous residues in the lac repressor, we have now predicted the structure of the portion of the repressor sugar-binding site which accommodates the galactosyl moiety. This prediction provides the first potential view of the inducer/anti-inducer site in the repressor protein.

Base Sequence↗
PubMedDate not supplied

Sulphate sequestered in the sulphate-binding protein of Salmonella typhimurium is bound solely by hydrogen bonds.

An important question in understanding substrate binding by proteins is how charged groups are stabilized in the absence of their solvation shell. We have addressed this question here by solving the structure of the sulphate-binding protein of Salmonella typhimurium with bound substrate at 2.0 A resolution. The results are remarkable in that the charged oxygen atoms of the sulphate molecule, which is buried and completely inaccessible to the solvent, are not stabilized by the formation of salt-bridges but by hydrogen bonds donated by specific residues of the protein. These hydrogen bonds are in turn coupled via peptide units to several resonating hydrogen bonding systems. These findings may be of general significance for the role of electrostatic interactions in protein structure and function.

Bacterial Proteins↗
PubMedDate not supplied

A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis.

The refined 1.9-A resolution structure of the periplasmic D-galactose-binding protein (GBP) reveals a calcium ion surrounded by seven ligands, all protein oxygen atoms. A nine-residue loop (amino-acid positions 134-142), which is preceded by a beta-turn and followed by a beta-strand, provides five ligands from every second residue. The last two ligands are supplied by the carboxylate group of Glu 205. The entire GBP Ca2+-binding site adopts a conformation very similar to the site in the 'helix-loop-helix' or 'EF-hand' unit commonly found in intracellular calcium-binding proteins, but without the two helices. Structural analyses have also uncovered the sugar-binding site some 30 A from the calcium and a site for interacting with the membrane-bound trg chemotactic signal transducer approximately 45 A from the calcium. Our results show that a common tight calcium binding site of ancient origin can be tethered to different secondary structures. They also provide the first demonstration of a metal-binding site in a protein which is involved in bacterial active transport and chemotaxis.

Amino Acid Sequence↗