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Biomedical subjects

R Nagaraj

Publications and source records attributed to R Nagaraj.

At least 19 recordsLinked to original sources
PubMed2005

Detection of peptides covalently modified with multiple fatty acids by MALDI-TOF mass spectrometry.

Analysis and characterization of membrane proteins and hydrophobic peptides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a considerable challenge because of their lower ionization efficiency. Detergents are used to solubilize hydrophobic peptides and proteins. However, in MALDI-MS, the presence of detergents can cause considerable loss of signal intensity. The extent of interference depends on the matrix/sample preparation method and experimental conditions. In the present study, we have analyzed the MALDI response of multiple fatty acylated peptides in the presence of the matrices alpha-cyano-4-hydroxy cinnamic acid (HCCA) and 2,5-dihydroxy benzoic acid (DHB). The effect of adding the nonionic detergent n-octylglucoside (OG) was also examined. The presence of OG facilitated detection of tetrapalmitoylated peptide, particularly when HCCA was used as the matrix. When DHB was used as the matrix, good signal intensity was observed in the absence of OG. Lower laser pulse rate in the linear mode of analysis resulted in good signal intensity for the tetrapalmitoylated peptide. Conditions for obtaining good signal intensities for dipalmitoylated and N-myristoyl peptides with both HCCA and DHB as matrices were also investigated.

Acylation↗
PubMed2004

Bacterial lipid modification of proteins for novel protein engineering applications.

Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited. Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use Escherichia coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end. We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change. Taking periplasmic Shigella apyrase as an example, its signal sequence was engineered to include a lipobox, an essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp. Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass spectrometric analysis. Substrate specificity and specific activity measurements indicated functional integrity after modification. In conclusion, a convenient protein engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully.

Amino Acid Sequence↗
PubMed2003

Cyclic homooligomers of furanoid sugar amino acids.

Cyclic homooligomers of mannose-derived furanoid sugar amino acid 1 [H-Maa(Bn(2))-OH] were synthesized by using BOP reagent in the presence of DIPEA under dilute conditions that converted the sugar amino acid monomer directly into its cyclic homooligomers 3a and 4a. The glucose-based sugar amino acid 2 [H-Gaa(Bn(2))-OH] under the same reaction conditions gave a bicyclic lactam 5a as the major product. Cyclic homooligomers of 2 were prepared by cyclizing their linear precursors 6 and 7 leading to the formation of cyclic peptides 8a and 9a, respectively. Conformational analysis by NMR and constrained MD studies revealed that all the cyclic products, 3, 4, 8, and 9, had symmetrical structures. The deprotected cyclic trimer of Maa 3b displayed a conformation in which all the C=O and the N-H bonds of the molecule point in opposite directions. In the deprotected cyclic tetramer of Maa 4b, the COs and NHs were in the plane of the ring with the former pointing to outside and the latter inside the ring. The structure of the cyclic Gaa dimer 8b displayed an unusual six-membered intramolecular hydrogen bond between NH(i)() --> C3-O(i)()(-)(1) and a syn orientation between the C2-H and CO. In this molecule, the C2-hydrogens and the COs can be seen on one side of the ring while the NHs point to the other side. Addition of the bicyclic lactam 5b resulted in the influx of Na(+) ions across the lipid bilayer leading to the dissipation of valinomycin-mediated K(+) diffusion potential.

Amino Acids, Neutral↗
PubMed2002

Effects of deleting a tripeptide sequence observed in muscular dystrophy patients on the conformation of synthetic peptides corresponding to the scaffolding domain of caveolin-3.

The caveolin-scaffolding domain (CSD) is a region in caveolin-1 and 3 that mediates interactions with signaling proteins. In some patients with limb-girdle muscular dystrophy, a three amino acid micro deletion in the CSD has been observed. The conformations and aggregation behavior of synthetic peptides, corresponding to the CSD of caveolin-3: DGVWKVSYTTFTVSKYWFY and the sequence where TFT (underlined in the native sequence) has been deleted, have been investigated. Circular dichroism spectra and molecular dynamics simulations indicate distinctive differences in the conformations of the native and mutant sequences. The extent of self-association in aqueous medium is also less pronounced in the case of the peptide with the micro deletion. It is likely that the structural changes arising as a result of TFT deletion distrupt oligomerization and consequently mistargeting and degradation.

Amino Acid Sequence↗
PubMed2002

NMR study of cyclic peptides with renin inhibitor activity.

Several cyclic analogues of renin inhibitors, based on Glu-D-Phe-Lys motif have been investigated by NMR spectroscopy and molecular dynamics calculations (MD). The 15 membered macrocycle, resulting from Glu and Lys side-chain cyclization, exhibits conformational preference. The structural evidence from NMR shows the presence of hydrogen bond between Lys NH and Glu side-chain carbonyl, resulting in a 10 membered pseudo beta-turn-like structure. The structure of the cyclic moiety is similar in all the peptides, which takes at least two conformations around Calpha-Cbeta in Glu side chain. The restrained MD calculations further support such observations and show that the macrocycle is fairly rigid, with two conformations about the Glu Calpha-Cbeta bond. The linear peptide appendages, which are essential for activity in cyclic peptides, show an extended structure in the beta-region of Ramchandran plot. These calculations also demonstrate that for the most active peptide, two major conformers each exist about the Calpha-CO bond of the Lys, D-Trp and Leu residues. In this peptide, the cyclic moiety presents a negatively charged surface formed due to the carbonyl oxygens, which are thus available to form hydrogen bonds with the receptor. The linear fragment presents further binding sites with a surface which has the hydrophobic side chains of D-Trp, Leu and D-Met on one side and carbonyls on the other side.

Circular Dichroism↗
PubMed2002

Antibacterial activities and conformations of bovine beta-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity.

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil beta-defensin BNBD-12, GPLSC(1)GRNGGVC(2)IPIRC(3) PVPMRQIGTC(4) FGRPVKC(5) C(6)RSW with disulfide connectivities C(1)-C(5), C(2)-C(4) and C(3)-C(6) and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt beta-hairpin or beta-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid beta-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in beta-defensins.

Amino Acid Sequence↗
PubMed2002

Antibacterial activities and conformations of synthetic alpha-defensin HNP-1 and analogs with one, two and three disulfide bridges.

Structure and biological activities of synthetic peptides corresponding to human alpha-defensin HNP-1, AC1YC2RIPAC3IAGERRYGTC4IYQGRLWAFC5C6 with the S-S connectivities: C1-C6, C2-C4, C3-C5, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP-1 yielded a peptide with S-S connectivities C1-C3, C2-C4 and C5-C6, and not with the S-S linkages as in naturally occurring HNP-1. Selective protection of cysteine sulfhydryls was necessary for the formation of S-S bridges as in native HNP-1. Likewise, oxidation of peptide encompassing the segment from C2 to C5, resulted in the S-S linkages C2-C3 and C4-C5 instead of the expected linkage C2-C4 and C3-C5. Antibacterial activities were observed for all peptides, irrespective of how the S-S bridges were linked. Linear peptides without S-S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S-S bridges do not form rigid beta-sheet structures in an aqueous environment. The spectrum of HNP-1 in an aqueous environment suggests the presence of a beta-hairpin conformation. In the presence of lipid vesicles, the S-S constrained peptides tend to adopt a beta-structure. Although the S-S connectivities observed in HNP-1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.

Amino Acid Sequence↗
PubMed2002

Host-defense antimicrobial peptides: importance of structure for activity.

Antimicrobial peptides are important components of innate immunity in species across the evolutionary scale. Unlike therapeutically used antibiotics, this class of peptides exert their activity by permeabilizing bacterial membranes. Despite the seemingly common mechanism of action, there is considerable variation in their primary structures, length and number of positive charges. Host-defense antimicrobial peptides have been the subject of extensive biophysical studies with a view at delineate structural requirements for activity. In this article, the structures of host defence antibacterial peptides and the structural requirements for activity are reviewed.

Amino Acid Sequence↗
PubMed2002

The therapeutic potential of host-defense antimicrobial peptides.

Species across the evolutionary scale from insects to mammals deploy peptides to counter microbial invaders. Although, there are considerable differences in the primary and secondary structures of these peptides, they exert their antimicrobial activity by common mechanisms ie. by membrane permeabilization. Extensive structure-function studies have been carried out on this class of antimicrobial peptides and 'designer' peptides have been generated which possess specific antimicrobial activity. In this article the physico-chemical properties and biological activities of host-defense peptides and 'designer' peptides would be reviewed with a view to explore whether they could be used as effective therapeutic agents.

Amino Acid Sequence↗
PubMed2001

Biological activities of retro and diastereo analogs of a 13-residue peptide with antimicrobial and hemolytic activities.

The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13-residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C-terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C-terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.

Amino Acid Sequence↗
PubMed2001

Structure-function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C-terminal end to a central position favors selective antibacterial activity.

Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC, is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide-bridged forms have relatively broad-spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKCAANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.

Amino Acid Motifs↗
PubMed2001

Effects of salt and denaturant on structure of the amino terminal alpha-helical segment of an antibacterial peptide dermaseptin and its binding to model membranes.

The amino terminal 1-18 domain of dermaseptin s is an important determinant of its structure as well as the antibacterial activity. A thorough investigation on the structure of the 18-residue peptide (D18) and its binding to model membranes in presence of salt and denaturant guanidinium chloride has been carried out. In presence of salt, there is an increase in the fraction of peptide molecules in helical conformation. In presence of the denaturant, D18 is unordered, but addition of the structure-promoting solvent trifluoroethanol results in a transition to the helical conformation. In presence of denaturant, the peptide is unordered, but binding to lipid vesicles is not abolished. Investigation of model membrane permeabilizing ability of the peptide in solutions containing various proportions of sodium chloride and guanidinium chloride indicates that vesicle permeabilization parallels extent of binding. The peptide thus binds to lipid vesicles in an unfolded state. Since the peptide has propensity to fold into a helical conformation, lipid induced transition to a helical structure occurs, followed by membrane permeabilization as a result of pore formation.

Amino Acid Sequence↗
PubMed2000

Tigerinins: novel antimicrobial peptides from the Indian frog Rana tigerina.

Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenaline-stimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form beta-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.

Amino Acid Sequence↗
PubMed2000

Synthesis and conformational studies of peptidomimetics containing furanoid sugar amino acids and a sugar diacid.

Furanoid sugar amino acids (1) were synthesized and used as dipeptide isosteres to induce interesting turn structures in small linear peptides. They belong to a new variety of designed hybrid structures that carry both amino and carboxyl groups on rigid furanose sugar rings. Four such molecules, 6-amino-2,5-anhydro-6-deoxy-D-gluconic acid (3, Gaa) and its mannonic (4, Maa), idonic (5, Iaa), and a 3,4-dideoxyidonic (6, ddIaa) congeners were synthesized. The synthesis followed a novel reaction path in which an intramolecular 5-exo S(N)2 opening of the hexose-derived terminal aziridine ring in 2 by the gamma-benzyloxy oxygen with concomitant debenzylation occurred during pyridinium dichromate oxidation of the primary delta-hydroxyl group to carboxyl function, leading to the formation of furanoid sugar amino acid frameworks in a single step. Incorporation of these furanoid sugar amino acids into Leu-enkephalin replacing its Gly-Gly portion gave analogues 8-11. Detailed structural analysis of these molecules by circular dichroism (CD) and various NMR techniques in combination with constrained molecular dynamics (MD) simulations revealed that two of these analogues, 8a and 10a, have folded conformations composed of an unusual nine-membered pseudo beta-turn-like structure with a strong intramolecular H-bond between LeuNH --> sugarC3-OH. This, in turn, brings the two aromatic rings of Tyr and Phe in close proximity, a prerequisite for biological activities of opioid peptides. The analgesic activities of 8a,b determined by mouse hot-plate and tail-clip methods were similar to that of Leu-enkephalin methyl ester. The syn disposition of the beta-hydroxycarboxyl motif on the sugar rings appears to be the driving force to nucleate the observed turn structures in some of these molecules (8 and 10). Repetition of the motif on both sides of a furanose ring resulted in a novel molecular design of sugar diacid, 2,5-anhydro-D-idaric acid (7, Idac). Bidirectional elongation of the diacid moieties of 7 with identical peptide strands led to the formation of a C2-symmetric reverse-turn mimetic 12 which displayed a very ordered structure consisting of identical intramolecular H-bonds at two ends between LeuNH --> sugar-OH, the same as in 8 and 10.

Amino Acids↗
PubMed2000

Combinatorial signaling in the specification of unique cell fates.

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Here, we demonstrate that together with the transcription factor Lozenge, the nuclear effectors of the EGFR and Notch signaling pathways directly regulate D-Pax2 transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of D-Pax2 expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. We expect that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication.

Animals↗
PubMed2000

Antibacterial and hemolytic activities of single tryptophan analogs of indolicidin.

The structure and biological activities of analogs of the bovine neutrophil antibacterial and hemolytic peptide indolicidin, ILPWKWPWWPWRR-amide, where one tryptophan at 4th, 8th, or 11th position has been retained and the others replaced by leucine, have been investigated. All the single tryptophan analogs exhibit antibacterial activity. However, unlike indolicidin, they do not lyse erythrocytes. Structure analysis by circular dichroism spectroscopy indicates that the analogs are unordered in aqueous medium and adopt beta-turn structures in trifluoroethanol and micelles. The tryptophan residues in indolicidin appear to be essential for hemolytic activity but not antibacterial activity. The nonspecific biological activities of indolicidin and specific antibacterial activity of single tryptophan analogs suggest that in short peptides, a motif composed of hydrophobic amino acids with the exception of tryptophan, interspaced with proline residues and cationic amino acids at the N or C termini would favor selective antibacterial activity.

Amino Acid Substitution↗
PubMed2000

Double-helical cyclic peptides: design, synthesis, and crystal structure of figure-eight mirror-image conformers of adamantane-constrained cystine-containing cyclic peptide cyclo (Adm-Cyst)(3).

A large number of macrocycles containing alternating repeats of cystine diOMe(-NH-CH(CO(2)Me)-CH(2)-S-)(2) and either a conformationally rigid aromatic/alicyclic moiety or a flexible polymethylene unit (X) in the cyclic backbone with ring size varying from 13- to 78-membered have been examined by spectral ((1)H NMR, FT-IR, CD) and X-ray crystallography studies for unusual conformational preferences. While (1)H NMR measurements indicated a turnlike conformation for all macrocycles, stabilized by intramolecular NH.CO hydrogen bonding, as also supported by FT-IR spectra in chloroform, convincing proof for beta-turn structures was provided by circular dichroism studies. Single-crystal X-ray studies on 39-membered cyclo (Adm-L-Cyst)(3) revealed a double-helical fold (figure-eight motif) for the macrocycle. Only a right-handed double helix was seen in the macrocycle constructed from L-cystine. The mirror-image macrocycle made up of D-cystine units exhibited a double helix with exactly the opposite screw sense, as expected. The enantiomeric figure-eights were stabilized by two intramolecular NH. CO hydrogen bonds and exhibited identical (1) H NMR and FT-IR spectra. The CD spectra of both isomers had a mirror-image relationship. The present results have clearly brought out the importance of cystine residues in inducing turn conformation that may be an important deciding factor for the adoption of topologically important structures by macrocycles containing multiple S-S linkages.

Crystallography, X-Ray↗