This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.104.044305v1 87/5/3504    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dzikovski, B. G. Articles by Freed, J. H. Search for Related Content PubMed PubMed Citation Articles by Dzikovski, B. G. Articles by Freed, J. H.

Spin-Labeled Gramicidin A: Channel Formation and Dissociation

Boris G. Dzikovski ^* , Petr P. Borbat ^* and Jack H. Freed ^*

^* Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York; and Center of Photochemistry, Russian Academy of Sciences, Moscow, Russia

Correspondence: Address reprint requests to Jack H. Freed, Cornell University, Dept. of Chemistry and Chemical Biology, B52 Baker Lab, Ithaca, NY 14853-1301. Tel.: 607-255-1301; E-mail: jhf{at}msc.cornell.edu.

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35°C (the gel to P_ß phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 Å, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the P_ß and L phases of DPPC (above 35°C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (0.4–1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.

This article has been cited by other articles:

				Y. E. Nesmelov, C. B. Karim, L. Song, P. G. Fajer, and D. D. Thomas Rotational Dynamics of Phospholamban Determined by Multifrequency Electron Paramagnetic Resonance Biophys. J., October 15, 2007; 93(8): 2805 - 2812. [Abstract] [Full Text] [PDF]