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J T Groves

Publications and source records attributed to J T Groves.

33 records · Page 2Linked to original sources
PubMed1996

Architecture and function of membrane proteins in planar supported bilayers: a study with photosynthetic reaction centers.

We present a simple and convenient method for creating fluid supported bilayers which contain oriented and functional photosynthetic reaction centers (RCs). The supported bilayers are prepared by fusion of proteoliposomes with a glass surface. The proteoliposomes are prepared by spontaneous insertion of RCs into preformed small, unilamellar vesicles. The RCs in these vesicles are shown to be oriented with the cytochrome c binding surface on the outside and the H-subunit facing inside. Upon fusion to glass surfaces, the RCs remain functional and highly oriented, with the cytochrome c binding surface exposed to the bulk solution. The RCs in the supported bilayers are at a surface density of order 10(11) RCs/cm2. The quality of the supported lipid bilayer is characterized by epifluorescence microscopy and the long-range lateral mobility of the lipids by fluorescence recovery after photobleaching. We demonstrate that homogeneous, fluid bilayers can be prepared over large areas (e.g., 1 cm2) of clean glass surfaces. The lipids in these supported bilayers are laterally mobile, and their diffusion coefficient agrees with values obtained in other fluid bilayer systems. This fluidity is unaffected by the presence of RCs; however, the RCs bearing a site-specific fluorescent label are immobile, despite retaining their charge separation and cytochrome c binding properties. We speculate that this results from interactions between the globular domain of the H-subunit and the glass substrate. Because of the unique spectroscopic and functional signatures associated with intact RCs, this system is one of the best characterized examples of a transmembrane protein in a supported bilayer at a nonbiological interface.

Lipid Bilayers↗
PubMed1996

Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.

Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.

Base Sequence↗
PubMed1996

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.

Amino Acid Sequence↗
PubMed1995

Electric field-induced concentration gradients in planar supported bilayers.

A simple method of generating electric field-induced concentration gradients in planar supported bilayers has been developed. Gradients of charged, fluorescently labeled probes were visualized by epifluorescence microscopy and could be observed at field strengths as low as 1 V/cm. Steady-state concentration gradients can be described by a simple competition between random diffusion and electric field-induced drift. A model based on this principle has been used to determine the diffusion coefficient of the fluorescent probes. This technique achieves a degree of electrical manipulation of supported bilayers that offers a variety of possibilities for the development of new molecular architectures and the study of biological membranes.

Biophysical Phenomena↗
PubMed1988

A unique deuterium/proton exchange during cytochrome P-450 mediated expoxidation of propene and butene.

A cytochrome P-450 LM2 reconstituted system mediated expoxidations of small terminal alkenes to the corresponding alkyloxiranes. This oxidation was accompanied by a stereoselective proton exchange of the E proton. Propene was oxidized in D2O to trans-3-deuterio-2-methyloxirane, and E-1-deuteriopropene was epoxidized in H2O to methyloxirane. Thus, oxidation occurred with complete exchange. The epoxidation of 1-butene in D2O generated a mixture of 86% ethyloxirane and 14% E-3-deuterio-2-ethyloxirane. Reaction of E-1-deuterio-1-butene produced 10-30% ethyloxirane and 70-90% trans-3-deuterio-2-ethyloxirane. Larger terminal alkenes in the homologous series did not undergo such an exchange. A mechanism involving formation of organoiron porphyrin intermediates is proposed. Stereoselective deprotonation by a basic amino acid residue producing an iron carbene complex is proposed as the mechanism of exchange with water.

Alkenes↗
PubMed1983

X-ray absorption spectroscopic studies of high valent iron porphyrins. Horseradish peroxidase compounds I and II and synthetic models.

X-ray absorption edge and extended x-ray absorption fine structure studies have been undertaken on resting (ferric) horseradish peroxidase, HRP compound I (HRP-I), HRP compound II (HRP-II), and several highly oxidized synthetic iron porphyrins that may have relevance as models for the iron site in horseradish peroxidase. The energies of the absorption edges are consistent with an Fe(IV) formulation for the highly oxidized species. The shapes of the absorption edges further support the assignment of HRP-I and one of the model compounds as Fe(IV)-porphyrin pi-cations. The edge shapes also demonstrate that the iron sites in the model porphyrins are not identical to the iron sites in the enzyme. Preliminary curve fitting analysis of the extended x-ray absorption fine structure clearly indicates the presence of two different nearest neighbor distances for the iron, both in HRP-I and HRP-II, as well as in two of the highly oxidized model porphyrins. These distances are interpreted as an iron-porphyrin nitrogen distance and as a short (approximately 1.6 A) iron-oxygen distance.

Electron Probe Microanalysis↗
PubMed1979

Electronic and steric factors in regioselective hydroxylation catalyzed by purified cytochrome P-450.

A reconstituted hydroxylation system consisting of electrophoretically homogeneous phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450 LM2), NADPH-cytochrome P-450 reductase, phospholipid, buffer, NADPH, and O2 was used to oxidize four cyclohexane derivatives: cyclohexene, methylcyclohexane, norcarane and norbornane. Cyclohexene gave only cyclohexene oxide and allylic cyclohexenol, while methylcyclohexane yielded all possible monohydric alcohols, but with 1 degrees:2 degrees:3 degrees ratios of 0.072:1:1.25. Norcarane yielded 2-norcaranol. While oxidation of norbornane produced exo-2- and endo-2-norborneols in a ratio of 3.4:1, replacement of all four exo-hydrogens by deuterium led to a reversal of the exo:endo ratio to 0.76:1. These and other observations are interpreted as evidence for a selective, hydrogen-abstracting enzyme-bound oxidant exhibiting a large intramolecular deuterium isotope effect. A transient substrate carbon radical is a probable intermediate in the hydroxylation process.

Animals↗