Structural Dynamics of Light-Driven Proton Pumps

Abstract

The physical properties of amorphous biomolecules are important to the texture and stability of low-moisture foods, the stability of pharmaceuticals, the permeability of edible films, and the viability of organisms during anhydrobiosis. Protein stability is often improved via the inclusion of small-molecule excipients during freeze-drying and organisms overproduce sugars such as sucrose or trehalose during anhydrobiosis. The effect on internal protein dynamics caused by substitution of a protein's surface water molecules with small sugar molecules is unclear. To explore this question, we have analyzed tryptophan phosphorescence decays of human serum albumin (HSA) in the dry amorphous solid state. Phosphorescence is an ideal approach, as the long-lived triplet state of tryptophan is sensitive to the long time-scale molecular motions of proteins in the dry state. Human serum albumin (HSA) was chosen because it contains a single, buried tryptophan residue and thus can provide information on the local dynamics of a specific site in the interior of the protein. Amorphous protein films were prepared by spreading concentrated solutions of HSA with and without sugar onto quartz slides, followed by rapid drying and extensive desiccation. Phosphorescence intensity decays were collected and fit with multiple exponential functions. From the average lifetime of these fits the rates of nonradiative decay (kNR) of the triplet state were calculated; kNR is dependent on the microviscosity of the site and is thus a measure of molecular mobility of the HSA tryptophan site. At all temperatures this measure of molecular mobility was lower in the films containing sucrose. Break-point analysis of a kNR Arrhenius plot revealed two temperature regimes with a transition occurring at the glass transition temperature of sucrose.

Conformational activation of visual rhodopsin in native disc membranes

“…1B), indicating that structural changes in photoactivated rhodopsin can be described by a single conformational state over this time window. This principal component was reminiscent of a TR-WAXS difference spectrum recorded after light activation of bacteriorhodopsin (24) (fig. S4).…”

“…Several complementary approaches exist to refine structural changes against difference WAXS data (24,25,27,(29)(30)(31)(32)(33). Unlike x-ray crystallography, however, broadly accepted standards for structural refinement have not yet emerged.…”

“…Every sample volume was exposed to either x-rays (laser off) or x-rays and laser (laser on) only once and then discarded. X-ray scattering data were integrated in rings and normalized (24). Difference TR-WAXS curves were retrieved for each time delay by subtracting the average of the flanking laser off cycles from the laser on recording.…”

“…Although the protein's x-ray scattering is relatively weak in the WAXS region, light-induced changes in the WAXS spectra can be measured very accurately. When the resting-state structure is known, protein structural changes can be refined against the difference WAXS signal (24,25,27,(29)(30)(31)(32)(33).…”

Overcoming barriers to membrane protein structure determination

“…Time-resolved Laue diffraction has been used to observe light-induced electron density changes in a photosynthetic reaction centre 88 , and time-resolved wide-angle X-ray scattering has provided low-resolution overviews of lightinduced helical movements with time in bacteriorhodopsin and proteorhodopsin 89 . Although…”