Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

Abstract

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X‐ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH‐values. The observed light‐induced intensity changes of in‐plane X‐ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X‐ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3–3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at ‐180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

“…5) that are still present at high Tris concentration and are also observable with more hydrophobic buffers such as imidazole. These pH-independent absorbance changes are attributed to a transient change in surface potential caused, e.g., by alterations in the tertiary structure of BR (10,13,14). Thus, fluorescein, which is located in the diffuse double layer, might enter another environment.…”

“…From the position of the chromophore retinal (5,6), bound to Lys-216, the location of the protonated nitrogen of the Schiff base that is part of the active center of this transport protein can be inferred. To correlate H+ ejection kinetics with the corresponding spectroscopic intermediate(s) of the BR photocycle (7,8), with protonation changes of amino acids (9)(10)(11)(12), and with alterations of the tertiary structure of the protein in the vicinity of the chromophore (13,14), H+ transfer was monitored with the optical pH indicator fluorescein covalently linked to the extracellular surface of BR, at Lys-129. Concomitant surface potential changes were reflected by absorption changes of both fluorescein and the potentiometric dye 4-{[2-(di-n-butylamino)-6-naphthyl]vinyl}-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS).…”

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family

“…Structural changes of prolines could indeed be detected with BR of H. halobium by Fourier transform infrared spectroscopy (18,41). It is possible that these structural changes of prolines are related to movements of part of the protein during the photocycle which could be detected by time-resolved X-ray diffraction (27).…”

Proposal to DOE Basic Energy Sciences: Ultrafast X-ray science facility at the Advanced Light Source

“…X-rays are ideal probes of atomic structure because they interact with core electronic levels that are closely bound to the atomic nucleus. A growing number of research groups around the world are applying ultrafast x-ray techniques to investigate structural dynamics in a variety of condensed matter systems [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], however, future progress in this field is substantially limited at present due to the lack of suitable short-pulse x-ray sources.…”

“…Femtosecond optical techniques have been widely applied to investigate ultrafast biological processes [2], and coherent vibrational motion has been observed to play a role in biological reactions such as the first step in vision [66][67][68], photosynthetic charge transfer [69], and photodissociation in heme proteins [73]. Of course quantitative structural information requires x-ray measurements, and to date x-ray techniques have been applied on time scales from milliseconds to nanoseconds to investigate structural dynamics in biological systems such as heme protein [21,104], bacteriorhodopsin [22], and photoactive yellow protein [12]. Extending structural measurements of biological systems to the femtosecond time scale is a significant challenge due to demanding x-ray source requirements, but it is nevertheless an important long-term goal.…”