Single-Particle Tracking Reveals Switching of the HIV Fusion Peptide between Two Diffusive Modes in Membranes

Fusion of the HIV membrane with that of a target T cell is an essential first step in the viral infection process. Here we describe single-particle tracking (SPT) studies of a 16-amino-acid peptide derived from the HIV fusion protein (FP₁₆), as it interacts with a supported lipid bilayer. FP₁₆ was found to spontaneously insert into and move within the bilayer with two different modes of diffusion, a fast mode with a diffusion coefficient typical of protein motion in membranes and a much slower one. We observed transitions between the two modes: slow peptides were found to speed up, and fast peptides could slow down. Hidden Markov model analysis was employed as a method for the identification of the two modes in single-molecule trajectories and analysis of their interconversion rates. Surprisingly, the diffusion coefficients of the two modes were found to depend differently on solution viscosity. Thus, whereas the fast diffusive mode behaved as predicted by the Saffman–Delbrück theory, the slow mode behaved according to the Stokes–Einstein relation. To further characterize the two diffusive modes, FP₁₆ molecules were studied in bilayers cooled through their liquid crystalline-to-gel phase transition. Our analysis suggested that the slow diffusive mode might originate from the formation of large objects, such as lipid domains or local protrusions, which are induced by the peptides and move together with them

Coupling and Decoupling of Rotational and Translational Diffusion of Proteins under Crowding Conditions

Molecular motion of biopolymers <i>in vivo</i> is known to be strongly influenced by the high concentration of organic matter inside cells, usually referred to as crowding conditions. To elucidate the effect of intermolecular interactions on Brownian motion of proteins, we performed ¹H pulsed-field gradient NMR and fluorescence correlation spectroscopy (FCS) experiments combined with small-angle X-ray scattering (SAXS) and viscosity measurements for three proteins, αB-crystalline (αBc), bovine serum albumin, and hen egg-white lysozyme (HEWL) in aqueous solution. Our results demonstrate that long-time translational diffusion quantitatively follows the expected increase of macro-viscosity upon increasing the protein concentration in all cases, while rotational diffusion as assessed by polarized FCS and previous multi-frequency ¹H NMR relaxometry experiments reveals protein-specific behavior spanning the full range between the limiting cases of full decoupling from (αBc) and full coupling to (HEWL) the macro-viscosity. SAXS was used to study the interactions between the proteins in solution, whereby it is shown that the three cases cover the range between a weakly interacting hard-sphere system (αBc) and screened Coulomb repulsion combined with short-range attraction (HEWL). Our results, as well as insights from the recent literature, suggest that the unusual rotational–translational coupling may be due to anisotropic interactions originating from hydrodynamic shape effects combined with high charge and possibly a patchy charge distribution