Active Membrane Fluctuations Studied by Micropipet Aspiration

We present a detailed analysis of the micropipet experiments recently reported in J-B. Manneville et al., Phys. Rev. Lett. 82, 4356--4359 (1999), including a derivation of the expected behaviour of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane, and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by the new expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range. [Submitted to Phys Rev E, 22 March 2001]Comment: 14 pages, 5 encapsulated Postscript figure

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions

Bacteriorhodopsin in ice accelerated proton transfer from the purple membrane surface

AbstractThe photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and in ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H+-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure