Structural Changes in the Photoactive Site of Proteorhodopsin during the Primary Photoreaction

Abstract

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

“…The protein samples for the low-temperature FTIR measurements were prepared as previously reported [20,21] using approximately 200 μg of the protein for each experiment. The samples were deposited on CaF 2 windows and dried under a slow stream of argon.…”

“…Samples were then rehydrated via the vapor phase and sealed in a sample cell with another CaF 2 window and mounted on a liquid nitrogen cryostat (ARS Cryo Helitran LT-3). Lowtemperature FTIR difference spectra were recorded using a protocol similar to that reported previously on green proteorhodopsin [21][22][23], BR [24,25], and other microbial rhodopsins [26,27]. For measurements at 80 K, the samples were first cooled in the dark from a lightadapted state at room temperature to avoid trapping of photointermediates.…”

“…Only the frequency of bands discussed in this paper are labeled in this and subsequent figures a slight upshift measured by resonance Raman spectroscopy (RRS data not shown). An empirical inverse relationship between the visible absorption maximum and the ethylenic stretching frequency has been established for rhodopsins [33] and microbial rhodopsins (see for example [21,26,34]), which indicates that such an upshift might correspond to a few nanometer blue-shift of the λ max of AR3 relative to BR at 80 K. The λ max of light-adapted AR3 reconstituted in E. coli polar lipids at room temperature was found to be near 570 nm (data not shown) but the UV-visible absorption at 80 K has not yet been measured.…”

Early Photocycle Structural Changes in a Bacteriorhodopsin Mutant Engineered to Transmit Photosensory Signals

“…Earlier, we reported structural changes in BR and SRII detected by means of low temperature FTIR spectroscopy (15)(16)(17)(18). In this study, the FTIR spectra of BR-T in the presence and absence of HtrII are compared with those of BR and SRII.…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…These studies were all conducted in collaboration with John Spudich, a leader in the microbial rhodopsin field at the University of Texas Health Science Center at Houston. They included FTIR difference studies along with RRS on halorhodopsin (HR) [197], sensory rhodopsins (SRI, SRII) from archaebacterial [29,31,34,38,184], fungal neurospora rhodopsin (NO) [30], anabaena sensory rhodopsin from cyanobacteria (ASR) [32], green and blue proteorhodopsin proton pumps from marine bacteria (PRs) [4,5,24,28,33,129,130]; archaerhodopsins-3 (AR3), a BR-like protein [56,216] and most recently channelrhodopsins (ChRs) from algae [173,174,176,250]. These proteins span a range of functions which are representative of diverse biomembranes process and include active anion transport (HRs), signal transduction (SRI, SRII and ASR), and light-gated ion channels (ChRs).…”