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R Radhakrishnan

Publications and source records attributed to R Radhakrishnan.

At least 55 records · Page 3Linked to original sources
PubMed1988

X-ray diffraction analysis of the inhibition of porcine pancreatic elastase by a peptidyl trifluoromethylketone.

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.

Animals↗
PubMed1988

Structure of native porcine pancreatic elastase at 1.65 A resolutions.

The structure of native porcine pancreatic elastase in 70% methanol has been refined using film data to 1.65 A resolution, R = 0.169. A total of 134 molecules of water (but no methanol) has been refined. This structure, because of its native state and modestly high resolution, serves as the basis for comparison with other elastase structures complexed with natural or synthetic ligands. Internal structured water occupies distinct regions. Two regions (IW1 and IW7) suggest a mechanism for equalizing 'hydrostatic pressure' related to ligand binding and release. A third region (IW4) forms part of a hydrogen-bonding network linking the catalytic Ser 195 O gamma with a remote (13.4 A) surface of the enzyme. A comparison with the structures of all known serine proteases reveals that a linkage of Ser O gamma to remote surface is conserved in all cases, suggesting that the accepted catalytic mechanism of serine proteases needs to be re-evaluated. One possible mechanism for base catalysis of Ser O gamma H proton extraction is presented.

Animals↗
PubMed1986

Structure of the product complex of acetyl-Ala-Pro-Ala with porcine pancreatic elastase at 1.65 A resolution.

A single crystal of porcine pancreatic elastase was mounted in a thin-walled capillary and allowed to react with acetyl-Ala-Pro-Ala-paranitroanalide. Diffraction data to 1.65 A resolution were measured and the isomorphous structure was solved from the difference Fourier map. The structure contains two surprises. Two molecules of the product: acetyl-Ala-Pro-Ala molecule are bound in the extended binding site. Both molecules are bound backwards with respect to the established mode of peptide binding.

Amino Acid Sequence↗
PubMed1985

Synthesis of phospholipids containing photoactivatable carbene precursors in the headgroups and their crosslinking with membrane proteins.

Many integral membrane enzymes require for their activity interactions with the polar headgroups of phospholipids, in addition to the hydrophobic interactions within the lipid bilayer. The interactions with the polar headgroups may have preferential or absolute specificity. To study such interactions, phospholipids have been synthesized which carry photoactivable moieties in their headgroups. Three types of phospholipids, PL-I, PL-II and PL-III, were synthesized. The synthetic phospholipids, PL-I and PL-II were able to reconstitute enzymatic activity of the membrane proteins which were studied. Covalent crosslinking between these phospholipids and the membrane proteins was demonstrated after photolysis of the reconstituted phospholipid-protein complexes.

Bacteriorhodopsins↗
PubMed1983

The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids.

Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-omega-(2-diazo-3,3,3-trifluropropionyloxy)lauroyl-sn- glycero-3-phosphocholine (PC-II). Rabbit liver cytochrome b5 was inserted into these vesicles spontaneously and the resulting vesicles containing the cytochrome b5 in the transferable form were photolyzed. Cytochrome b5 containing covalently cross-linked phospholipids was isolated by Sephadex LH-60 column chromatography using ethanol/formic acid as the solvent. Of the total radioactivity, 4.6% (PC-I) or 11.3% (PC-II) was linked to the protein; of the former, up to 51% was base-labile, while in the latter, 22% was base-labile. The sites of cross-linking of PC-I to the protein were investigated by fragmentation with trypsin, Staphylococcus aureas V8 protease, CNBr, and o-iodosobenzoic acid followed by Sephadex LH-60 chromatography and Edman sequencing (solid phase) of the appropriate fragments. The distribution of cross-linking was broad (Ser-104 to Met-130), showing a bell-shaped pattern with a significant peak at Ser-118. The labeling pattern is consistent with the previously proposed loop-back model for the membranous segment in the transferable form of cytochrome b5.

Animals↗
PubMed1983

The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids. II. The nontransferable form.

Rabbit liver cytochrome b5 was reconstituted into vesicles containing dipalmitoyl-sn-glycero-3-phosphocholine and the radioactively labeled photoactivatable 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) by the cholate-gel filtration method. The cytochrome inserted predominantly in the nontransferable form (Preparation I). In addition, reconstituted vesicles containing asymmetrically distributed radioactively labeled PC-I were prepared by the use of the phosphatidylcholine transfer protein. This preparation (Preparation II) contained [14C]PC-I in the outer leaflet and [3H]PC-I predominantly in the inner leaflet. Following photolysis of the labeled vesicle preparations, the purified cross-linked products were analyzed for the distribution of the sites of cross-links. In Preparation I, a broad distribution (Asn-105 to Met-130) was found, similar to that observed with the transferable form. On the other hand, the peptide fragments obtained from the asymmetrically labeled preparation II showed 14C/3H ratios which decreased in going from Asn-105 to Met-130. We conclude that the membrane-embedded segment in the nontransferable form of cytochrome b5 spans the bilayer.

Amino Acid Sequence↗
PubMed1983

Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.

The lipid binding site of the phosphatidylcholine transfer protein from bovine liver has been investigated by use of phosphatidylcholine analogs which carry a diazirinophenoxy group linked to the omega-carbon of either the sn-2-[1-14C]hexanoyl (PC I) or sn-2-[1-14C]undecanoyl chain (PC II). Photolysis of the PC I(PC II)-transfer protein complex resulted in a covalent coupling of 30-40% of the label to the protein as shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Upon mild alkaline treatment of the photolysed complex the protein containing covalently coupled 14C-label was separated from the noncoupled 14C-label by gel permeation chromatography. The 14C-labeled protein was degraded with protease from Staphylococcus aureus, trypsin and cyanogen bromide and specific 14C-labeled peptides were sequenced by automated Edman degradation. Major sites of coupling shown by release of radioactivity were identified as Tyr54 and the peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177. Both PC I and PC II coupled extensively to Tyr54 (90% and 50% of total labeling, respectively). The remainder of the radioactivity was released from the peptide Val171-Asp177 with a distinct difference in in the pattern of release depending on whether PC I or PC II were used. Thus, coupling occurred preferentially to Tyr175 and Asp177 with PC I while Val171 and Met173 were labeled preferentially with PC II. This shift in coupling is compatible with an increase of 0.6 nm for the sn-2-fatty-acyl chains of PC I and II, assuming that the peptide Val171-Asp177 has adopted the strongly predicted beta-strand configuration. These data have been interpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.

Amino Acid Sequence↗
PubMed1982

Orientation of retinal in bacteriorhodopsin as studied by cross-linking using a photosensitive analog of retinal.

The photosensitive m-diazirinophenyl analog of retinal (Fig. 1, II) bound to bacterio-opsin at Lys-216 and regenerated a chromophore with lambda max at 470 nm. Photolysis of the complex at 365 nm resulted in covalent cross-linking of the retinal analog to the bacterio-opsin in greater 30% yield. Investigation of the sites of cross-linking between the 3H-labeled retinal analog and the protein showed the peptide fragment (amino acid residues 190-248) to be the main radioactively labeled product. Stepwise Edman degradation showed Ser-193 and Glu-194 to be the predominant sites of cross-linking. These results show that the chromophore in bacteriorhodopsin is inclined towards helix 6 and towards the exterior of the cell. These data also provide information on the approximate angle that the chromophore makes with the plane of the membrane and they require a modification of the current secondary structure model for bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1982

The transmembrane domain of glycophorin A as studied by cross-linking using photoactivatable phospholipids.

Glycophorin A, the major sialoglycoprotein of the human erythrocyte, consists of a NH2-terminal carbohydrate-rich region exposed to the outside, a hydrophobic region which forms a transmembrane bridge, and a COOH-terminal hydrophilic region extending into the cytoplasm. With the aim of further defining the membrane-embedded region, the protein has been reconstituted into vesicles formed from dimyristoylphosphatidylcholine and phospholipids containing photosensitive carbene precursors. The photosensitive groups were incorporated either at the omega-position of sn-2 fatty acyl chain or the polar head group of lecithins. Following photolysis, covalent cross-linking (1-2%) of the photoactivatable phospholipids to the protein was demonstrated. Degradation and sequence analysis showed that in the case of phospholipids containing photoactivatable groups in the fatty acyl chains most of the covalent cross-linking involved the carboxyl group of Glu-70. Therefore, the latter residue must be within the bilayer. This conclusion was supported by the reaction of the membrane-permeant [14C] dicyclohexylcarbodiimide with glycophorin reconstituted into vesicles. The same residue was labeled. Photolysis of glycophorin vesicles containing phospholipids with photolabels in the polar head group gave products in which the cross-links were present in peptide fragments (residues 62-81 and 82-96). These results define the probable boundaries of the membrane-embedded segment of glycophorin A. Corresponding experiments with erythrocyte ghosts gave similar results.

Cross-Linking Reagents↗
PubMed1981

Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins.

In an approach to the study of phospholipid-protein interactions in biological membranes, the photoactivable fatty acids, omega-(m-azidophenoxy)-undecanoic acid (I) and omega-(m-diazirinophenoxy)-hexadecanoic acid (II), were incorporated biosynthetically into the phospholipids of the Escherichia coli fatty acid auxotroph, strain K1060-B5. The extent of incorporation of the two fatty acids was 43% and 21%, respectively, of the total fatty acid content of the phospholipids. Membrane vesicles prepared from cells grown on the fatty acid supplements and [32P]H3PO4 were irradiated at suitable wavelengths to generate the reactive nitrene or carbene intermediates. Subsequent analysis of solubilized membrane proteins by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis indicated cross-linking between radioactive phospholipids and an number of proteins. A corresponding experiment with cells grown on oleic acid showed only trace amounts of covalently cross-linked phospholipid-protein adducts. While the extent of cross-linking in vesicles from cells grown on I was only 3 times the background level observed for oleic acid-grown cells, cells grown on II showed 30 times this amount. The present results, together with the previously observed nonreactivity of the nitrene generated from I to undergo C-H insertion, show that the use of carbene precursors such as II is promising for chemical analysis of specific phospholipid-protein interactions in bacterial membranes under biologically meaningful conditions.

Cell Membrane↗
PubMed1981

Light-driven proton translocation by bacteriorhodopsin reconstituted with the phenyl analog of retinal.

The properties of bacteriorhodopsin in which the natural cofactor, retinal (Fig. 1, I), has been replaced by the synthetic analog, phenyl retinal (Fig. 1, II), have been studied. Phenyl retinal binds at the same site as retinal and supports light-dependent transmembrane proton translocation in phospholipid vesicles that contain bacterio-opsin. This result allows us to rule out proton abstraction from the beta-ionone ring of retinal as a step in the catalytic cycle. Furthermore, phenyl retinal, a planar molecule, binds to the apomembrane more rapidly than retinal, which suggests that the rate-determining step in the binding of retinal itself may be the attainment of a planar molecular configuration. The new chromophore undergoes a pH-dependent spectral shift that is not observed in the native membrane.

Bacteriorhodopsins↗
PubMed1981

Site of attachment of retinal in bacteriorhodopsin.

The identity of the lysine residue in bacteriorhodopsin to which the chromophore, retinal, is attached as a Schiff's base has been reinvestigated. Retinal is now shown to be linked to lysine-216 and not lysine-41, as had been concluded previously. The retinal in purple membrane was replaced by [15-3H]retinal, the Schiff's base linkage was reduced with NaBH4 while the sample was illuminated, and the resulting [retinyl-3H]bacterio-opsin was cleaved with CNBr. The radioactivity was present exclusively in the COOH-terminal peptide (amino acids 210--248). Sequence analysis showed that the [3H]retinal was attached to lysine-216. The same site was labeled when purple membrane was reduced with NaBH4 in the light at pH greater than 10 or at pH 8 and when membranes modified with ether or solubilized with hexadecyltrimethylammonium bromide were reduced in the dark. Our finding places of Schiff's base linkage close to the midpoint of the putative membrane-spanning alpha-helix that is directly connected to the COOH terminus of bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1980

Phospholipids containing photoactivable groups in studies of biological membranes.

Photoactivable carbene precursors, aryl diazirines and trifluorodiazopropionates, were incorporated synthetically into the omega-positions of fatty acids, which were used to synthesize phospholipids. Extensive intermolecular C--H insertion reactions were demonstrated by photolysis of liposomes prepared from the above phospholipids. Structural analysis of the cross-linked products showed that the predominant sites of cross-linking were in the expected positions within the bilayer. Studies on the topography of a number of membrane proteins using the above phospholipids were initiated. Cross-linking of the photoactivable phospholipids to membrane-embedded proteins, glycophorin A, cytochrome b5, and gramicidin A, was demonstrated.

Animals↗