MOLECULAR FLEXIBILITY DEMONSTRATED BY PARAMAGNETIC ENHANCEMENTS OF NUCLEAR-RELAXATION - APPLICATION TO ALAMETHICIN - A VOLTAGE-GATED PEPTIDE CHANNEL

A nitroxide spin label attached to the C-terminus of the channel formi ng peptide alamethicin produces an enhancement of the nuclear spin-lat tice relaxation rates of peptide protons as a result of both intermole cular and intramolecular magnetic dipole-dipole interactions. The inte rmolecular contribution provides evidence that alamethicin monomers co llide preferentially in a C-terminal-to-N-terminal configuration in me thanol. From the intramolecular paramagnetic enhancement of nuclear sp in-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alam ethicin, and cannot be accounted for by motion in the bonds that attac h the nitroxide to the peptide. In addition, the differences between d istances deduced from the nuclear spin relaxation and the distances se en in the crystal structure increase toward the N-terminal end of the peptide, The simplest explanation for these data is that the alamethic in backbone suffers large structural fluctuations that yield shorter e ffective distances between the C-terminus and positions along the back bone. This finding can be interpreted in terms of a molecular mechanis m for the voltage-gating of the alamethicin channel. When the distance s between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r(-5 ), and distances calculated using the Solomon-Bloembergen equations ma y more nearly represent a distance of closest approach than a time ave rage distance. Therefore, the use of paramagnetic centers such as spin labels or metal ions with long electron T-1 values provides a distanc e measurement that reflects a dynamically averaged structure where the averaging process heavily weights short distances. The results of suc h measurements, when combined with other structural information, may p rovide particularly clear evidence for the magnitude of structural flu ctuations involving distances greater than 10 Angstrom.