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Biomedical subjects

C E Schutt

Publications and source records attributed to C E Schutt.

At least 37 records · Page 2Linked to original sources
PubMed1995

The effect on actin ATPase of phalloidin and tetramethylrhodamine phalloidin.

Actin polymerization has been studied in the absence of excess nucleotide. Using G-actin ATP monomers, it was shown that mechanical shearing stimulates ATP hydrolysis. The procedures used enabled the detection of differential effects of phalloidin and tetramethylrhodamine-phalloidin, on the P(i)-release step of the actin ATPase. It is concluded that tetramethylrhodamine, in contrast to phalloidin, accelerates P(i)-release from actin filaments.

Actins↗
PubMed1995

Structural studies on the ribbon-to-helix transition in profilin: actin crystals.

Knowledge of the structure of actin in its various conformational states is important for understanding the diverse motile activities carried out by eukaryotic cells. Profilin:actin crystals provide a unique system for studying conformational states of actin, because they exhibit a high degree of polymorphism in response to environmental conditions while maintaining crystalline order. A preliminary comparison of two states of profilin:beta-actin crystals shows that crystal polymorphism involves movements of actin subdomains at hinge points homologous to those found in hexokinase, a protein whose polypeptide fold is related to actin. The homology of the hinge points in actin to those in hexokinase suggests that actin subdomain movements in profilin:beta-actin crystals have functional significance. We discuss how these movements could be related to structural transitions between states of filamentous actin in muscle contraction.

Actins↗
PubMed1994

Crystallization and structure determination of bovine profilin at 2.0 A resolution.

Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1994

Isomorphous binding of mercury-substituted thiosaccharides to pertussis toxin crystals yields crystallographic phases.

An isomorphous derivative of pertussis toxin crystals was prepared using a 2-alpha-mercuric analog of N-acetyl neuraminic acid in a method analogous to the use of inhibitors labelled with heavy atoms to solve crystal structures of enzymes. This derivative exploits the specific binding between pertussis toxin and terminal sialic acid residues on receptor glycoproteins. Difference Patterson maps yielded heavy-atom sites which refined with good statistics, indicating that the protein probably does not undergo a conformational change on receptor binding. Mercuric analogs of other monosaccharides should be easily obtainable using the same synthetic strategy, suggesting a general method for derivatizing crystals of carbohydrate-binding proteins.

Journal Article↗
PubMed1994

Structural aspects of actin-binding proteins.

The three-dimensional structures of myosin subfragment 1 (S1), gelsolin segment 1 complexed with alpha-actin, villin fragment 14T, Acanthamoeba profilin-I, and bovine profilin complexed with beta-actin were completed last year. Together, they expand our understanding of the structural organization of actin-binding proteins. In addition, the segment 1 and bovine profilin complexes provide atomic-level descriptions of their interfaces with actin.

Actins↗
PubMed1993

The structure of crystalline profilin-beta-actin.

The three-dimensional structure of bovine profilin-beta-actin has been solved to 2.55 A resolution by X-ray crystallography. There are several significant local changes in the structure of beta-actin compared with alpha-actin as well as an overall 5 degrees rotation between its two major domains. Actin molecules in the crystal are organized into ribbons through intermolecular contacts like those found in oligomeric protein assemblies. Profilin forms two extensive contacts with the actin ribbon, one of which appears to correspond to the solution contact in vitro.

Actins↗
PubMed1993

Mutagenesis of human profilin locates its poly(L-proline)-binding site to a hydrophobic patch of aromatic amino acids.

The actin-binding protein, profilin, contains a src-homology (SH) 3-like fold (Schutt, C.E. et al., submitted), and its tight interaction with poly(L-proline) is reminiscent of the binding activity exhibited by SH3-domains. Here we demonstrate that replacements of aromatic amino acids in a hydrophobic patch on the surface of the profilin molecule abolish its poly(L-proline)-binding capacity. However, the location of this hydrophobic patch is found in another region of the molecule than that displaying structural similarities with SH3 domains.

Amino Acids↗
PubMed1993

Mutations in beta-actin: influence on polymer formation and on interactions with myosin and profilin.

Two beta-actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine-374 replaced with serine (C374S), as well as the wild-type beta-actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro 'motility assay'. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild-type actins.

Actins↗
PubMed1993

A new perspective on muscle contraction.

Recent experimental findings suggest that the myosin cross-bridge theory may no longer be adequate to account for certain basic facts concerning muscle contraction. A newly-proposed mechanism based on length changes in actin filaments might be the basis for a simpler explanation for how the free energy of ATP hydrolysis can be transduced into work by muscle fibers.

Actins↗
PubMed1992

Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase.

CheB, the methylesterase of chemotactic bacteria, catalyzes the hydrolysis of glutamyl-methyl esters in bacterial chemoreceptor proteins. The two cysteines predicted by the amino acid sequence of CheB were replaced by alanine residues. The resulting mutants, Cys207-Ala, Cys309-Ala and a double cysteine mutant Cys207-Ala/Cys309-Ala, retained methylesterase activity, indicating that sulfhydryls are not crucial for CheB mediated catalysis. A homology search revealed a conserved serine active-site region between residues 162 and 166 which is homologous to the active-site region of acetylcholine esterases, suggesting that Ser164 of CheB is the active-site nucleophile. Oligonucleotide-directed mutagenesis was used to change the serine to a cysteine. This Ser164-Cys mutant had less than 2% of the wild-type activity. Unlike the serine proteinases which utilize a 'catalytic triad' mechanism, CheB does not have the conserved histidine and aspartic acid residues located in positions N-terminal to the active-site serine. In addition, CheB is not labeled with di-isopropylfluorophosphate, a potent inhibitor of other serine hydrolases. A novel mechanism is proposed for CheB involving substrate-assisted catalysis to account for these apparent anomalies.

Amino Acid Sequence↗
PubMed1992

Actin as the generator of tension during muscle contraction.

We propose that the key structural feature in the conversion of chemical free energy into mechanical work by actomyosin is a myosin-induced change in the length of the actin filament. As reported earlier, there is evidence that helical actin filaments can untwist into ribbons having an increased intersubunit repeat. Regular patterns of actomyosin interactions arise when ribbons are aligned with myosin thick filaments, because the repeat distance of the myosin lattice (429 A) is an integral multiple of the subunit repeat in the ribbon (35.7 A). This commensurability property of the actomyosin lattice leads to a simple mechanism for controlling the sequence of events in chemical-mechanical transduction. A role for tropomyosin in transmitting the forces developed by actomyosin is proposed. In this paper, we describe how these transduction principles provide the basis for a theory of muscle contraction.

Actins↗
PubMed1991

Crystallization and preliminary X-ray analysis of Gc, the vitamin D-binding protein in serum.

The vitamin D-binding protein, Gc, was purified from human serum and crystallized using the hanging-drop method. The best crystals were grown from 28% polyethylene glycol 400 in 50 mM-sodium acetate at pH 4.8. These crystals diffract to 3.4 A and the observed diffraction is consistent with orthorhombic space groups P4(1) and P4(3). The unit cell parameters were determined to be a = b = 135.5 A and c = 75.6 A.

Chromatography, Ion Exchange↗
PubMed1991

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor.

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.

Amino Acids↗
PubMed1990

Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis.

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.

Bordetella pertussis↗