Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid-labeled peptides.

The attractive interaction between basic protein domains and membranes containing acidic lipids is critical to the membrane attachment of many proteins involved in cell signaling. In this study, a series of charged model peptides containing lysine, phenylalanine, and the spin-labeled amino acid tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) were synthesized, and electron paramagnetic resonance (EPR) spectroscopy was used to determine their position on the membrane interface and free energy of binding. When membrane-bound, peptides containing only lysine and TOAC assume an equilibrium position within the aqueous double layer at a distance of approximately 5 A from the membrane interface, a result that is consistent with recent computational work. Substitution of two or more lysine residues by phenylalanine dramatically slows the backbone diffusion of these peptides and shifts their equilibrium position by 13-15 A so that the backbone lies several angstroms below the level of the lipid phosphate. These results are consistent with the hypothesis that the position and free energy of basic peptides when bound to membranes are determined by a long-range Coulombic attraction, the hydrophobic effect, and a short-range desolvation force. The differences in binding free energy within this set of charged peptides is not well accounted for by the simple addition of free energies based upon accepted side chain partition free energies, a result that appears to be in part due to differences in membrane localization of these peptides.

Differences between the photocycles of halorhodopsin and the acid purple form of bacteriorhodopsin analyzed with millisecond time-resolved FTIR spectroscopy.

At pH 1, bacteriorhodopsin (bR) is thought to function as a halide ion pump, in contrast to its biological function as a proton pump at neutral pH. Despite the apparent similarity in function between this 'acid purple' form of bR and the native form of halorhodopsin (hR), their FTIR difference spectra measured ca. 5 ms after photolysis are significantly different. The most striking difference is the appearance of a positive band at 1753 cm-1 and a negative band at 1732 cm-1 in the bRacid purple difference spectrum. These and other spectral features are similar, but not identical, to those of the bR-->O difference spectrum measured at neutral pH. The structure of the bRacid purple longest-lived product therefore corresponds more closely to the O photoproduct of the bR proton-pumping photocycle, rather than the hL photoproduct seen on a similar time scale in the hR photocycle. The 1753- and 1732-cm-1 bands are largely unaffected by the D212N mutation, but both appear to lose a portion of their intensities with either the D85N or D96N mutation. Thus Asp-85 and -96 likely undergo substantial changes in hydrogen-bonding environment during the halide-pumping cycle of bRacid purple. Our FTIR results deepen the distinctions between the hR and bR photocycles. The mechanism of chloride pumping in hR has been thought not to involve protonation or hydrogen bonding changes of carboxylic acid groups. In bRacid purple, however, it seems likely that at least one carboxylic acid might play an important role in the mechanism of chloride pumping, leading to an increase in thermodynamic or kinetic stabilization of the O intermediate.