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R M Stroud

Publications and source records attributed to R M Stroud.

At least 127 records · Page 7Linked to original sources
PubMed1987

The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis.

The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.

Animals↗
PubMed1987

Atomic structure of thymidylate synthase: target for rational drug design.

The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding.

Amino Acid Sequence↗
PubMed1986

Location of terbium binding sites on acetylcholine receptor-enriched membranes.

Acetylcholine receptor-enriched membranes bind 45 terbium cations per receptor. The Tb(III) X-ray scattering factor changes by as much as 30% over a 50 eV range about the L3 absorption edge. We exploit these changes to modulate the contribution of these ions to the X-ray diffraction pattern of oriented receptor-enriched membranes by varying the incident X-ray energy. Difference Fourier analysis of the meridional diffraction amplitudes at two X-ray energies revealed six localized regions of Tb(III) density across the membrane. Most significant is the finding of 18 Tb(III) ions near the entrance and 11 ions near the exit of the ion channel as well as 4 or 5 Tb(III) ions localized in the channel itself. This evidence strongly suggests the presence of anionic carboxylate side-chains on the channel lining.

Animals↗
PubMed1986

Food-induced (allergic) arthritis. Inflammatory arthritis exacerbated by milk.

Suggestive, but largely unproven, observations have associated arthritis with environmental antigens, including foods. We studied a patient with inflammatory arthritis in a prospective, "blinded," controlled fashion to determine whether her symptoms were associated with food sensitivities. This 52-year-old white woman with 11 years of class I, stage I, active disease, had symptomatic exacerbations allegedly associated with meat, milk, and beans. We observed an increase in symptoms following an unblinded food challenge and then studied her in our clinical research unit. On her normal diet for 6 days, she averaged 30 minutes of morning stiffness, 9 tender joints, 3 swollen joints, 87% subjective assessment (100% = best possible), and 89% examiner assessment. While she was fasting (3 days) or taking Vivonex (2 days), we noted no morning stiffness, tender joint score of 1, swollen joint score of 0, and assessments of 100% (P less than 0.05 versus normal diet). She was then nourished with Vivonex for 33 days without difficulty and challenged in a blinded fashion at mealtimes with lyophilized foods placed into opaque capsules. Four milk challenges (equivalent to greater than or equal to 8 ounces per meal) produced up to 30 minutes of morning stiffness, 14 tender joints, 4 swollen joints, subjective assessment of 85%, and objective assessment of 80% (P less than 0.05 versus fasting-Vivonex), peaking 24-48 hours postchallenge. Placebo and other foods (lettuce and carrots) were without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Cation binding sites on the projected structure of bacteriorhodopsin.

Divalent cations are involved in the function of bacteriorhodopsin (bR) as a light-driven proton pump. If cations are removed from purple membranes they become blue. Divalent cations such as Ca2+ or Pb2+ or trivalent ions, can be stoichiometrically titrated back on to these deionized membranes. The color transitions as a function of ion concentration for Ca2+ or Pb2+ are precisely comparable and indicate that approximately three stoichiometric equivalents of cations are required to effect the color transition (Kimura et al., 1984). We found four main partially occupied binding sites for cations at a stoichiometric ratio of 3 Pb2+/bR. We localized the binding sites for Pb2+ using x-ray diffraction of membranes reconstituted with 1, 2, and 3 equivalents of Pb2+ per bR. The site of highest affinity is located on helix 7. At 2 Pb2+/bR, sites on helix 6 and between helix 2 and 3 are occupied. At 3 Pb2+/bR a fourth site above helix 3 is occupied.

Bacteriorhodopsins↗
PubMed1985

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum.

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.

Amino Acid Sequence↗
PubMed1985

Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.

Amino Acid Sequence↗
PubMed1984

Location of an extrinsic label in the primary and tertiary structure of bacteriorhodopsin.

We located a heavy metal label, mercurilated phenylglyoxal, in both the primary sequence and in the tertiary structure of bacteriorhodopsin. This label modified arginines 225 and 227, which are on the COOH-terminal helix (G). In the projected electron potential difference map, the major site is close to the central inner helix. From this result we conclude that helix 1 could not be the COOH-terminal helix G. We tested the multiple isomorphous replacement method for obtaining phases for purple membrane by electron diffraction.

Amino Acid Sequence↗
PubMed1984

Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor.

Fourier analysis of the hydrophobicities of the acetylcholine receptor subunit sequences reveals regions of amphipathic secondary structure. Prediction of a consensus secondary structure based on this analysis and on an empirical prediction method leads to a testable hypothesis about how the ion channel is formed and might function. Knowledge of the three-dimensional structure of acetylcholine receptors is consistent with features of the model proposed and provides some constraints.

Amino Acid Sequence↗
PubMed1984

Subunit secondary structure in filamentous viruses: predictions and observations.

The algorithm of Garnier, Osguthorpe and Robson (J. Mol. Biol. 120, 97-120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, If1, IKe, Pf1, Xf and Pf3. For subunits of Class I virions (fd, If1, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pf1, Xf, Pf3), the algorithm consistently predicts a predominance of beta structure, which is compatible with the demonstrated facility for conversion of Class II subunits from alpha-helix to beta-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321-356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman (Adv. Enzym. 47, 45-148, 45-148). Therefore, both predictive and experimental methods indicate a distinction between Class I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform alpha-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.

Algorithms↗
PubMed1983

Subunit organization and structure of an acetylcholine receptor.

We have learned the positions of the alpha-subunits around the AChR rosette and the location of the toxin on the synaptic crest. A charge/hydrophobic character map of the 40 A X 30 A receptor surface that binds alpha-bungarotoxin has been constructed. A beta-structure domain surrounds the agonist binding site on the alpha-subunits, as predicted by amphipathic Fourier sequence analysis. The ion channel may be constructed from five amphipathic helices, which insert into the bilayer as the alpha2 beta gamma delta subunits come together. They form a water-filled channel on one side and interface with hydrophobic helices in each subunit on the other.

Amino Acid Sequence↗
PubMed1982

Structure and function of an acetylcholine receptor.

Structural analysis of an acetylcholine receptor from Torpedo californica leads to a three-dimensional model in which a "monomeric" receptor is shown to contain subunits arranged around a central ionophoretic channel, which in turn traverses the entire 110 A length of the molecule. The receptor extends approximately 15 A on the cytoplasmic side, 55 A on the synaptic side of the membrane. The alpha-bungarotoxin/agonist binding site is found to be approximately 55 A from the entrance to the central gated ion channel. A hypothesis for the mechanism of AcChR is presented which takes into account the structural and kinetic data, which is testable, and which serves as a focus for future studies on the agonist-induced structure change in AcChR.

Animals↗
PubMed1982

Linking regions between helices in bacteriorhodopsin revealed.

Three-dimensional electron-microscopic structural analysis requires the combination of many different tilted views of the same specimen. The relative difficulty of tilting the sample to high angles >60 degrees without introducing severe distortion due to different focal distances across the specimen entails that the observable range of electron diffraction data is often limited to this range of angles. Thus, it is generally not possible to observe the diffraction maxima that lie within the conical region of reciprocal space around the direction perpendicular to the electron microscope grid. The absence of data in this region leads to a predictable distortion in the object, and for +/-60 degrees tilting makes the resolution essentially twice as bad in the direction perpendicular to the grid as it is for the in-plane image. Constrained density map modification and refinement methods can significantly reduce these effects. A method has been developed, tested on model cases, and applied to the electron-microscopic structure determination of bacteriorhodopsin in order to visualize the location of linking regions between helices. Electron-microscopic structural analysis of bacteriorhodopsin (Henderson and Unwin. 1975 Nature [Lond.] 257:28-32.) showed that the molecule consists of seven rods of density each nearly spanning the lipid bilayer. Owing to the distortion introduced by the missing conical region of reciprocal space data, no density was visible for the polypeptide segments linking the alpha-helices. Density in the refined maps indicates the location of at least five of the extrahelical segments of the polypeptide. The total number of possible ways of interconnecting the helices is reduced from 7! (5,040) to the five most consistent possibilities without recourse to other considerations. In addition, the density for the helical regions is more uniform and cylindrical throughout their length, and the length of the helices increases from 35 to 45 A, close to the membrane thickness of 49 A obtained for membranes dried in vacuo. Only three of the five structures consistent with the location of observed linkers place the seventh helix, onto which the chromophore can be attached by reduction in the light, at a position consistent with the main peak for deuterated retinal in the structure, as derived from neutron diffraction analysis. Two of these models are also consistent with the possible location of some of the reduced chromophore on helix B, at lys 40/41 after reduction in the dark, as well as lys 216 on helix G.

Bacteriorhodopsins↗