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K U Prasad

Publications and source records attributed to K U Prasad.

At least 19 recordsLinked to original sources
PubMed1995

The determination of binding constants of micellar-packaged gramicidin A by 13C-and 23Na-NMR.

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30 degrees C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+, and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same ffmperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.

Amino Acid Sequence↗
PubMed1993

Self-assembly of bioelastomeric structures from solutions: mean-field critical behavior and Flory-Huggins free energy of interactions.

Elastic and quasi-elastic light scattering studies were performed on aqueous solutions of poly(Val-Pro-Gly-Gly), a representative synthetic bioelastomer that differs from the previously studied poly(Val-Pro-Gly-Val-Gly) by the deletion of the hydrophobic Val in position four. When the spinodal line was approached from the region of thermodynamic stability, the intensity of light scattered by fluctuations, and the related lifetime and correlation length, were observed to diverge with mean-field critical exponents for both systems. Fitting of the experimental data allowed determining the spinodal and binodal (coexistence) lines that characterize the phase diagrams of the two systems, and it also allowed a quantitative sorting out of the enthalpic and entropic contributions to the Flory-Huggins interaction parameters. The contribution of valine is derived by comparison of the two cases. This can be viewed as sorting out the effect of a modulation of the solute. The same approach may allow sorting out the entropic and enthalpic effect of modulations of the solvent by cosolutes (or by cosolvents). This could be of particular interest in the case of small osmolytes, affording important adaptive roles in nature, at the cost of very limited changes in genetic information. Finally, the suggestion is further supported that statistical fluctuations of anomalous amplitude, such as those occurring in proximity of the spinodal line, have a role in promoting the process of self-assembly of extended supramolecular structures. On the practical side, the present approach appears useful in the design of novel synthetic model systems for bioelastomers.

Amino Acid Sequence↗
PubMed1991

Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based polytetra-, polypenta-, and polynonapeptides.

Differential scanning calorimetry studies of the effect of NaCl on protein-based polymer self-assembly has been carried out on six elastin-based synthetic sequential polypeptides--i.e., the polypentapeptide (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n and (L-Val1-L-Pro2-L-Ala3-L-Val4-Gly5)n; the polytetrapeptide (L-Val1-L-Pro2-Gly3-Gly4)n and its more hydrophobic analogue (L-Ile1-L-Pro2-Gly3-Gly4)n; and the polynonapeptide (a pentatetra hybrid), (L-Val1-L-Pro2-Gly3-L-Val4-Gly5-L-Val6-L-Pro7-Gly8-Gly9++ +)n. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order of the polypentapeptide, as the temperature is raised from below to above that of the transition, has been repeatedly observed using different physical characterizations. In the present experiments, it is observed that the transition temperatures of the polypeptides studied are linearly dependent on NaCl concentration. The molar effectiveness of NaCl in shifting the transition temperature delta Tm/[N], is about 14 degrees C/[N], with the dependence on peptide hydrophobicity being fairly small. Interestingly, however, the delta delta Q/[N] does depend on the hydrophobicity of a polypeptide.

Amino Acid Sequence↗
PubMed1990

Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues.

Differential scanning calorimetry studies have been carried out on the sequential polypeptide of elastin, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n, abbreviated as PPP, and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Leu1-PPP, and (L-Ile1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Ile1-PPP Consistent with inverse temperature transitions, the temperatures of the transitions for which maximum heat absorption occurs are inversely proportional to the hydrophobicities of the polypentapeptides (31 degrees C for PPP, 16 degrees C for Leu1-PPP, and 12 degrees C for Ile1-PPP), and the endothermic heats of the transitions are small and increase with increasing hydrophobicity, i.e., 1.2, 2.9, and 3.0 kcal/mol pentamer for PPP, Leu1-PPP, and Ile1-PPP, respectively. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order, as the temperature is raised above that of the transition, has been repeatedly observed using different physical characterizations. Furthermore, the studies demonstrated identical conformations for PPP and Il21-PPP above and below the transition. Both heats and temperatures of the transitions vary with hydrophobicity, but not in simple proportionality.

Amino Acid Sequence↗
PubMed1990

Conformation of a cyclic decapeptide analog of a repeat pentapeptide sequence of elastin: cyclo-bis(valyl-prolyl-alanyl-valyl-glycyl).

The conformation of a cyclic decapeptide analog of a repeat sequence of elastin has been determined in the crystalline state using X-ray crystallographic techniques. Tetragonal crystals were grown from a solution of the decapeptide in water; space group P4(2)2(1)2, a = 19.439(2) & c = 13.602(1) A, with four formula units (C40H66N10O10.4H2O) per unit cell. The cyclic decapeptide in the crystal exhibits exact twofold symmetry. The asymmetric unit contains one pentapeptide and two water molecules for a total of 32 nonhydrogen atoms. The structure has been determined by the application of direct methods and refined by full-matrix least squares to an R index of 0.053 for 2272 reflections with intensities greater than 2 sigma(I). The backbone conformation of the asymmetric pentapeptide can be described as consisting of a double beta bend of Type III-I. The Type III turn has Pro (phi = -59.3 degrees, psi = -26.8 degrees) and Ala (phi = -65.9 degrees, psi = -23.1 degrees) at the corners while Type I turn has Ala (phi = -65.9 degrees, psi = -23.1 degrees) and Val (phi = -98.9 degrees, psi = 8.3 degrees) as the corner residues. The cyclic decapeptide has two such double bends linked together by Gly-Val bridges.

Amino Acid Sequence↗
PubMed1989

A mismatch between the length of gramicidin and the lipid acyl chains is a prerequisite for HII phase formation in phosphatidylcholine model membranes.

Previously it was shown that gramicidin can induce HII phase formation in diacylphosphatidylcholine model membranes only when the lipid acyl chain length exceeds 16 carbon atoms (Van Echteld, C.J.A., De Kruijff, B., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1982) Biochim. Biophys. Acta 692, 126-138). Using 31P-NMR and small angle X-ray diffraction we now demonstrate that upon increasing the length of gramicidin, the peptide loses its ability to induce HII phase formation in di-C18:1c-PC but not in the longer chained di-C22:1c-PC. It is concluded that a mismatch in length between gramicidin and the lipid acyl chains, when the latter would provide excess bilayer thickness, is a prerequisite for HII phase formation in phosphatidylcholine model membranes.

Gramicidin↗
PubMed1989

Two-dimensional proton NMR studies on poly(VPGVG) and its cyclic conformational correlate, cyclo(VPGVG)3.

Two-dimensional nuclear Overhauser enhancement (2D NOESY) data are reported for the polypentapeptide of elastin, poly(VPGVG), and the cyclopentadecapeptide, cyclo(VPGVG)3. In both, the repeating type II Pro2-Gly3 beta-turn can be derived from the NOE data, providing confirmation of many previous studies. In addition, other through-space connectivities are detailed that also compare favorably with previously determined crystal and solution structures for cyclo(VPGVG)3. Also, near identical data for the cyclopentadecapeptide and the polypentapeptide demonstrate the cyclic conformation-linear (helical) conformational correlate relationship between the two molecules. The 2D NOESY experiment is seen to be an effective means of establishing the presence or absence of a conformational relationship between a cyclic repeating sequence and its higher molecular weight linear counterpart. This is an approach of substantial practical value when developing the conformation of sequential polypeptides and when attempting to identify the presence of the conformation of a repeating peptide sequence within a more complex primary structure. Having established the basic conformational relationship between a cyclic conformation and its linear helical counterpart, cross peaks present in the linear helical structure that are not present in the cyclic conformational correlate can provide information on the interactions between adjacent turns of the helix. In this connection, a Val gamma CH3 in equilibrium Pro beta CH2 interaction is reported that can be the basis for determining the number of pentamers per turn of helix once it is determined whether it is dominantly the Val1 or Val4 gamma CH3 that is interacting with the Pro2 beta CH2.

Elastin↗
PubMed1989

Elastin repeat peptides as chemoattractants for bovine aortic endothelial cells.

Cultured bovine aortic endothelial cells migrate toward a concentration gradient of repeating elastin peptides, specifically the repeating nonamers Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro and Gly-Leu-Gly-Val-Gly-Ala-Gly-Val-Pro and the repeating hexamer Val-Gly-Val-Ala-Pro-Gly. Dose-response experiments demonstrate that the peak of activity occurs at 8 x 10(-8) M for the nonapeptides and 1 x 10(-8) M for the hexapeptide. Checkerboard assays establish that the movement is chemotaxis and not chemokinesis. Because of the concentration difference in the responsiveness between the nonapeptide and the hexapeptide, the cells can differentiate between the two types of repeats. The positive control for the chemotaxis studies was fibronectin.

Amino Acid Sequence↗
PubMed1989

Synthesis of 4% Glu-containing Val1 and Ile1-polypentapeptides: model protein systems for demonstrating mechanochemical coupling.

The synthesis of 4% Glu-polypentapeptide (PPP) (i.e., 4 Glu residues per 100 amino acid residues) and 4% Glu-Ile1-PPP, in which Val1 is substituted by a more hydrophobic Ile residue, is carried out by copolymerizing the p-nitrophenyl-active esters of GE(OMe)GVP and GE(OMe)GIP with their parent pentamers GVGVP and GVGIP in 1:4 ratios, respectively. After removal of the methyl ester on the side chain of Glu, these polymers exhibited a remarkable pH dependence of the temperature for their inverse temperature transitions, which are followed as turbidity development at 300 nm. On gamma-irradiation crosslinking, the elastomeric bands obtained exhibited a pH-mediated contraction and relaxation. Thus, for the first time, mechanochemical coupling is demonstrated in a synthetic polypeptide system. That the basic mechanism involves the hydrophobic effect (chemical modulation of an inverse temperature transition) and not ion-ion electrostatic repulsion is also discussed.

Glutamates↗
PubMed1989

Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin.

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.

Crystallography↗
PubMed1988

Nucleation and accretion of bioelastomeric fibers at biological temperatures and low concentrations.

Quasi-elastic light scattering (QELS) studies are reported, which address the early stages of aggregation of the polypentamer poly(VPGVG). This reflects the major primary structural feature of native elastin. The study is focused on the region of the phase diagram which in both its temperature and concentration range is closest to the state of affairs occurring in the course of bioelastogenesis by progressive synthesis of the precursor protein. Results here reported allow for the first time a self-consistent view of the physics of elastogenesis, and specify the role of the region of metastability and of that of instability of the phase diagram in the non-chaotic, orderly formation of elastomeric fibers.

Biopolymers↗
PubMed1988

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes.

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1988

pK shift of functional group in mechanochemical coupling due to hydrophobic effect: evidence for an apolar-polar repulsion free energy in water.

In the sequential polypeptide poly[4(VPGVG),(VPGEG)] and its more hydrophobic analog poly[4(IPGVG),(IPGEG)] when the material is gamma-irradiation cross-linked to form an elastomeric matrix, mechanochemical coupling occurs on changing the pH, that is, motion and mechanical work are achieved by a change in proton chemical potential. The temperature dependence of aggregation at different pH values in phosphate buffered saline demonstrates the pK to be shifted approximately one pH unit higher for the more hydrophobic sequential polypeptide. The pH dependence of contraction or relaxation for each elastomer shows a similar shift. Data are reviewed and 2D-NMR data are presented which argue that the pK shift is not due to different conformations of the polypentapeptides. Specifically it is proposed that there exist a competition between carboxylates and hydrophobic side chains for mutually incompatible water structures; this results in an apolar-polar repulsion free energy in water with the difference in free energy reflecting the difference in the lle and Val hydrophobicities.

Elastin↗
PubMed1988

Chemotaxis of fibroblasts toward nonapeptide of elastin.

Bovine ligamentum fibroblasts, which produce elastin, migrate towards a positive chemical gradient of human platelet-derived growth factor and of the tropoelastin repeat hexapeptide Val-Gly-Val-Ala-Pro-Gly, as previously shown. They are also responsive to two permutations of a nonapeptide that repeats in tropoelastin, i.e., Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly and Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro. Concentration curves and checkerboard assays prove that the nonapeptides are chemoattractants. The component pentapeptide, Gly-Phe-Gly-Val-Gly, is chemotactic, while the component tetrapeptide Ala-Gly-Val-Pro is not. The hexapeptide competitively suppresses the nonapeptide chemotaxis suggesting the involvement of a common cell receptor. The results support the concept that elastin has multiple cell recognition sites as measured by the chemotactic response and that among the hydrophobic repeating sequences of elastin chemotacticity is selectively and multiply localized.

Animals↗
PubMed1988

A synthetic polypentapeptide of elastin for initiating calcification.

A polypentapeptide (PPP) of tropoelastin having a repeating amino acid sequence of (Val-Pro-Gly-Val-Gly)n was evaluated for its potential to initiate in vivo calcification and to enhance bone formation in nonhealing calvarial wounds (8.0 mm) in 396 adult Walter Reed rats. There were four configurations of the PPP (molecular weight range of 50-100K dalton) consisting of 1-dry PPP; 2-coacervate PPP; 3-gamma irradiated, cross-linked PPP; 4-calcified, gamma irradiated, cross-linked PPP. These four iterations plus a control group of animals constituted the five treatment classes that were evaluated at days 1, 3, 7, 21, 42, and 147. Seventy two rats were used for each treatment and 36 rats for the control. Following euthanatization, specimens were placed into 70% ethanol, embedded in polymethyl methacrylate, sectioned at 3.5 micrometers, and alternating sections were stained with Masson-Goldner trichrome and von Kossa stains. Histomorphometric analysis was accomplished using a Zeiss Universal microscope (250 X) and Videoplan Image Analysis System to evaluate five random histologic fields from margin to margin of the craniotomy. Trabecular bony volume and area of calcification islands were quantitated. A Student's t test for unpaired data to determine treatment differences (within the same temporal groups) revealed that there was no significant difference between treatments and control for trabecular bony volume; however, there was a significant difference between experimentals and control for calcification islands (P less than 0.05) such that calcifications islands for the experimentals were greater than the control. There was not a significant difference between experimental treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Mechanochemical coupling in synthetic polypeptides by modulation of an inverse temperature transition.

For the polypentapeptide of elastin, (L-Val-L-Pro-Gly-L-Val-Gly)n, and appropriate analogs when suitably cross-linked, it has been previously demonstrated that development of elastomeric force at fixed length and length changes at fixed load occur as the result of an inverse temperature transition, with the temperature of the transition being inversely dependent on the hydrophobicity of the polypeptide. This suggests that at fixed temperature a chemical means of reversibly changing the hydrophobicity could be used for mechanochemical coupling. Evidence for this mechanism of mechanochemical coupling is given here with a 4%-Glu-polypentapeptide, in which the valine in position 4 is replaced in 1 out of 5 pentamers by a glutamic acid residue. Before cross-linking, the temperature for aggregation of 4%-Glu-polypentapeptide is remarkably sensitive to pH, shifting from 25 degrees C at pH 2 to 70 degrees C at pH 7.4 in phosphate-buffered saline (PBS). At 37 degrees C, the cross-linked 4%-Glu-polypentapeptide matrix in PBS undergoes a pH-modulated contraction and relaxation with a change from pH 4.3 to 3.3 and back. The mean distance between carboxylates at pH 4.3 in the elastomeric matrix is greater than 40 A, twice the mean distance between negatively charged species in PBS. Accordingly, charge-charge repulsion is expected to make little or no contribution to the coupling. Mechanochemical coupling is demonstrated at fixed load by monitoring pH dependence of length and at constant length by monitoring pH dependence of force. To our knowledge, this is the first demonstration of mechanochemical coupling in a synthetic polypeptide and the first system to provide a test of the recent proposal that chemical modulation of an inverse temperature transition can be a mechanism for mechanochemical coupling. It is suggested that phosphorylation and dephosphorylation may modulate structure and forces in proteins by locally shifting the temperatures of inverse temperature transitions.

Amino Acid Sequence↗
PubMed1987

On the mechanism of channel-length dependence of gramicidin single-channel conductance.

Single-channel conductance data on four different gramicidin channel lengths demonstrate that conductance magnitude is neither inversely dependent on the square of the channel length nor on the image force arising from differences in the extent of lipid dimpling (Jordan and Vayl (1985) Biochim. Biophys. Acta 818, 416-420). Rather the conductance differences are consistent with the decreased off-rate constant for the singly occupied state as the ionic radius decreases from that of cesium ion to sodium ion coupled with the decreased probability of the doubly occupied channel due to increased ion-ion repulsion as the channel is shortened (Urry et al. (1984) Biochim. Biophys. Acta 774, 115-119).

Cesium↗