Tina L. Trapane

Is the Gramicidin A Transmembrane Channel Single-Stranded or Double-Stranded Helix? A Simple Unequivocal Determination

Thallium ion-induced carbonyl carbon chemical shifts were compared for all of the L-residue-peptide carbonyl carbons of the gramicidin A transmembrane channel. Molecular structures were deduced by using the argument that helically equivalent and equally proximal carbonyls would exhibit essentially equivalent ion-induced chemical shifts. The transmembrane channel was found to be a head-to-head dimer with the structure of a left-handed, single-stranded β-helix.

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

SummaryMalonyl gramicidin is incorporated into lysolecithin micelles in a manner which satisfies a number of previously demonstrated criteria for the formation of the transmembrane channel structure. By means of sodium-23 nuclear magnetic resonance, two binding sites are observed: a tight site and a weak site with binding constants of approximately 100m−1 and 1m−1, respectively. In addition, off-rate constants from the two sites were estimated from NMR analyses to bekofft≃3×105/sec andkoffw≃2×107/sec giving, with the binding constants, the on-rate constants,kont≃3×107/msec andkonw≃2×107/m sec.Five different multiple occupancy models with NMR-restricted energy profiles were considered for the purpose of calculating single-channel currents as a function of voltage and concentration utilizing the four NMR-derived rate constants (and an NMR-limit placed on a fifth rate constant for intrachannel ion translocation) in combination with Eyring rate theory for the introduction of voltage dependence.Using the X-ray diffraction results of Koeppe et al. (1979) for limiting the positions of the tight sites, the two-site model and a three-site model in which the weak sites occur after the tight site is filled were found to satisfactorily calculate the experimental currents (also reported here) and to fit the experimental currents extraordinarily well when the experimentally derived values were allowed to vary to a least squares best fit. Surprisingly the “best fit” values differed by only about a factor of two from the NMR-derived values, a variation that is well within the estimated experimental error of the rate constants.These results demonstrate the utility of ion nuclear magnetic resonance to determine rate constants relevant to transport through the gramicidin channel and of the Eyring rate theory to introduce voltage dependence.

Test of the librational entropy mechanism of elasticity of the polypentapeptide of elastin. Effect of introducing a methyl group at residue 5

The polypentapeptide analogue, (Val1-Pro2-Gly3-Val4-Ala5)n, has been synthesized to test the proposed librational entropy mechanism of elasticity which is based on the polypentapeptide of elastin, (Val1-Pro2-Gly3-Val4-Gly5)n. The mechanism utilizes the motional capacities of the Val4-Gly5-Val1 segment of the elastin polypentapeptide in a β-spiral conformation in which the segment is suspended between β-turns. Proton and carbon-13 nuclear magnetic resonance spectroscopies have been used to verify purity and to demonstrate that the Pro2-Gly3β-turn is retained in the Ala5 analogue. In addition, the temperature profiles for aggregation are found to be similar for both polypentapeptides. Aggregation of the Ala5 analogue, however, so immobilizes the polypeptide as to give no carbon-13 nuclear magnetic resonance spectrum, whereas a broadened spectrum is observed for the aggregated state of the polypentapeptide of elastin. Scanning electron micrographs are used to compare the aggregated states of the two polymers. On settling, the polypentapeptide of elastin forms a smooth-appearing sheet whereas the addition of a methyl group at residue 5 results in a granular precipitate. In contrast to the elastomeric polypentapeptide of elastin, the Ala5 analogue, on cross-linking with γ-irradiation, simply fragments when stress/strain studies are attempted. Thus the introduction of a methyl group at residue 5 allows common conformational features to exist but destroys the elasticity in a manner consistent with the proposed librational entropy mechanism.The effects of this and other analogues of the polypentapeptide of elastin are discussed in terms of accessible configurations, and it is argued that by means of the β-spiral conformation the polypentapeptide of elastin provides a unique configurational entropy which is utilized to produce a new type of entropic elastomer.

Chapter 4 Ion Interactions with the Gramicidin A Transmembrane Channel: Cesium-133 and Calcium-43 NMR Studies

Publisher Summary This chapter illustrates the capacity to determine meaningful rate constants by means of NMR relaxation studies of spin 7/2 cesium- 133 and allow these approaches to be considered for spin 7/2 calcium-43. Background NMR data are presented for calcium-43, e.g., CaCI 2 concentration dependence and temperature dependence of the longitudinal relaxation time, TI, in D,O, in the presence of phosphatidylcholine lipid and in the presence of phosphatidylcholine lipid plus gramicidin A channels and the transverse relaxation time, T 2 for 100 m M and 1 M CaCI 2 , in the presence and absence of channels. These data allow determination of the off-rate constant for calcium ions leaving the channel binding site to be of the order of 5 x 10 7 /sec. With the binding constant demonstrated here to be about l/M for the site at the mouth of the gramicidin A channel and with the experimental off-rate constant, it becomes apparent that the lack of a calcium ion current is due to a high central barrier arising from the large repulsive image force which occurs when a divalent charge is separated from the lipid dielectric constant by no more than a single layer of polypeptide backbone.

Nuclear magnetic resonance and conformational energy calculations of repeat peptides of tropoelastin: conformational characterization of the cyclododecapeptide

N.m.r. data obtained in chloroform–dimethyl sulphoxide solutions and conformational-energy calculations are reported which deduce the preferred conformation of the cyclic dodecapeptide, cyclo-(L-Ala1-L-Pro2-Gly3-L-Val4-Gly5-L-Val6)2, having the hexapeptide repeat sequence of tropoelastin. The non-equivalence of the Gly α-CH2 protons and the similarity of α-carbon relaxation times indicate that the molecule is quite rigid. The 1H n.m.r. spectrum of the molecule resembles that of a hexamer, indicating that the molecule possesses two-fold symmetry on the n.m.r. time scale. The possible ranges of backbone torsion angles except ψ(Ala1) are derived from α-CH–NH coupling constants, geminal coupling constants, and nuclear Overhauser effects. From temperature-dependence studies of the peptide NH chemical shift and from the coupling constants, secondary structural features of the molecule are obtained. The valyl α-CH–β-CH coupling constants show that the Val4 side chain is in a gauche conformation, while the Val6 side chain is in the trans-conformation. A static wire model is developed using these data and containing the following solution-derived hydrogen bonds: CO(1)⋯ HN(4)(β-turn), NH(1)⋯ OC(4) and NH(6)⋯ OC(4)(bifurcated 14- and 17-membered rings), and NH(3)⋯ OC(5)(γ-turn). In vacuo conformational-energy calculations are performed to obtain several minimum-energy conformations. The theoretical calculations utilize the Go–Scheraga method for cyclization with exact two-fold symmetry. Only one of the low-energy structures so obtained is consistent with all the experimental data, and this structure is quite similar to the static wire model based on the n.m.r. data.