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IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH

Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion trans- port and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, atten- uated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.

Contains fulltext : 154849.pdf (Publisher’s version ) (Open Access) Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.

Contains fulltext : 174447.pdf (Publisher’s version ) (Closed access) A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet-visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C horizontal lineO stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 --> Gln and Ser97 --> Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 --> Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.

Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US and Western world. Despite increased screening and advances in treatment, the mortality rate (ca. 50,000/year) and high national health-care burden for CRC are likely to remain high unless an effective non-invasive screening test for CRC is instituted for a large segment of the population. Blood-based protein biomarkers hold great promise for early disease diagnosis and personalized medicine; yet robust and reproducible multiplexing platforms and methodologies have lagged behind their genomic counterparts.Here, we report the development of a novel, multiplexed, hybrid immunoassay for CRC that is formatted on barcoded VeraCode™ micro-beads, which have until now only been used for genomic assays. The method combines a sandwich immunoassay format for detection of serum protein biomarkers with an antigen assay for autoantibody detection. The serum protein biomarkers CEA and GDF15 as well as autoantibodies to the p53 tumor associated antigen (TAA) were used to exemplify the method. This multiplex biomarker panel was configured to run on Illumina's holographically barcoded VeraCode™ micro-bead platform, which is capable of measuring hundreds of analytes simultaneously in a single well from small volumes of blood (

Optogenetics relies on the expression of specific microbial rhodopsins in the neuronal plasma membrane. Most notably, this includes channelrhodopsins, which when heterologously expressed in neurons function as light-gated cation channels. Recently, a new class of microbial rhodopsins, termed anion channel rhodopsins (ACRs), has been discovered. These proteins function as efficient light-activated channels strictly selective for anions. They exclude the flow of protons and other cations and cause hyperpolarization of the membrane potential in neurons by allowing the inward flow of chloride ions. In this study, confocal near-infrared resonance Raman spectroscopy (RRS) along with hydrogen/deuterium exchange, retinal analogue substitution, and site-directed mutagenesis were used to study the retinal structure as well as its interactions with the protein in the unphotolyzed state of an ACR from Guillardia theta (GtACR1). These measurements reveal that (i) the retinal chromophore exists as an all-trans configuration with a protonated Schiff base (PSB) very similar to that of bacteriorhodopsin (BR), (ii) the chromophore RRS spectrum is insensitive to changes in pH from 3 to 11, whereas above this pH the Schiff base (SB) is deprotonated, (iii) when Ser97, the homologue to Asp85 in BR, is replaced with a Glu, it remains in a neutral form (i.e., as a carboxylic acid) but is deprotonated at higher pH to form a blue-shifted species, (iv) Asp234, the homologue of the protonated retinylidene SB counterion Asp212 in BR, does not serve as the primary counteranion for the protonated SB, and (v) substitution of Glu68 with an Gln increases the pH at which SB deprotonation is observed. These results suggest that Glu68 and Asp234 located near the SB exist in a neutral state in unphotolyzed GtACR1 and indicate that other unidentified negative charges stabilize the protonated state of the GtACR1 SB.

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600 cm( - 1), which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000 cm( - 1) assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

Microbial rhodopsins are an important class of light-activated transmembrane proteins whose function is typically studied on bulk samples. Here we apply photochromic fluorescence resonance energy transfer (pcFRET) to investigate the dynamics of these proteins with sensitivity approaching the single-molecule limit. The brightness of a covalently linked organic fluorophore is modulated by changes in the absorption spectrum of the endogenous retinal chromophore that occur as the molecule undergoes a light-activated photocycle. We studied the photocycles of blue-absorbing proteorhodopsin (BPR) and sensory rhodopsin II (SRII). Clusters of 2–3 molecules of SRII clearly showed a light-induced photocycle. Single molecules of SRII showed a photocycle upon signal averaging over several illumination cycles.

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Proteorhodopsins (PRs) are members of the microbial rhodopsin family. Since their initial discovery in Monterey Bay (1), over 4000 different variants have been discovered using environmental genome sequencing techniques (2–4). It is estimated that PR containing bacteria account for 13% of the microorganism species in the ocean photic zone (5). The widespread distribution of PR in worldwide oceanic waters indicates that PR phototrophy is a significant oceanic microbial process (5, 6). Similar to BR and other microbial rhodopsins, PRs consist of 7 transmembrane alpha-helices containing a retinylidene chromophore covalently bound to a lysine (Lys231) via a protonated Schiff base (1). Light absorption by PR results in an all-trans to 13-cis isomerization of the retinal which then drives a series of reaction steps that result in a proton being pumped from the cytoplasm to the extracellular medium (7, 8). There are two major subgroups of proteorhodopsins characterized by their visible absorption maxima. The green-absorbing proteorhodopsin (GPR) subgroup absorbs maximally at ~520 nm and function as a light driven proton pump similar to bacteriorhodopsin (BR) (1, 7–9). A second subgroup called blue-absorbing proteorhodopsin (BPR), absorbs near 490 nm (6). The different variants of PRs are thought to have evolved through Darwinian selection (10). One difference between GPR and BPR which was found to be a key determinant of their different properties is the identity of residue 105 (The numbering scheme for BPR is offset by one from GPR due to an extra glycine at residue 2 in BPR. Throughout, when referring to the amino acid positions in proteorhodopsins, the GPR numbering system is used). In GPR residue 105 is a leucine while in BPR it is a glutamine (11–14). In a related paper, we show using resonance Raman spectroscopy that this residue is involved in tuning the visible absorption via interaction with the Schiff base and the C13 methyl of the retinal chromophore (15). BPR also pumps protons; however it has a 10-fold slower photocycle than GPR. It has been suggested that the slower photocycle may indicate that BPR functions primarily as a photoregulator rather than an energy harvesting pigment (14, 16, 17). Fourier transform infrared (FTIR) difference spectroscopy has been successfully used to elucidate many of the details of the proton transport mechanism in BR (18–21) and GPR (7, 8, 22–26) as well to characterize the mechanisms of a variety of other microbial rhodopsins (27, 28). This study examines the initial events in the photocycle of BPR using static low-temperature FITR difference spectroscopy. The 80 K light induced difference spectra reflect the formation of a red shifted K-like photoproduct similar to the K-intermediates in GPR and light-adapted BR. Furthermore, the C=C and C-C retinal chromophore ethylenic and fingerprint stretching modes show that the primary photoreaction involves an all-trans → 13-cis isomerization of the retinal chromophore, although the difference spectrum also may exhibit small contributions from a 13-cis photocycle. Several prominent bands near 1700 cm−1 are assigned to structural changes of Asn230 and Gln105 side chains on the basis of site-directed mutations indicating that these residues are perturbed in the earliest events of the BPR photocycle. In addition, a pair of bands at 1551 (−) and 1537 (+) cm−1 are assigned to an amide II downshift on the basis of total-15N isotope labeling. Evidence is presented on the basis of assignment of bands to water molecule vibrations with H218O that there are two different sites for water molecules near the active site in BPR and GPR, respectively. In the Q105L mutant of BPR, which has a GPR-like visible absorption spectrum, both waters are present.

Proteorhodopsins are a recently discovered class of microbial rhodopsins, ubiquitous in marine bacteria. Over 4000 variants have thus far been discovered, distributed throughout the oceans of the world. Most variants fall into one of two major groups, green- or blue-absorbing proteorhodopsin (GPR and BPR, respectively), on the basis of both the visible absorption maxima (530 versus 490 nm) and photocycle kinetics ( approximately 20 versus approximately 200 ms). For a well-studied pair, these differences appear to be largely determined by the identity of a single residue at position 105 (leucine/GPR and glutamine/BPR). We find using a combination of visible and infrared spectroscopy that a second difference is the protonation state of a glutamic acid residue located at position 142 on the extracellular side of helix D. In BPR, Glu142 (the GPR numbering system is used) is deprotonated and can act as an alternate proton acceptor, thus explaining the earlier observations that neutralization of the Schiff base counterion, Asp97, does not block the formation of the M intermediate. In contrast, Glu142 in GPR is protonated and cannot act in this state as an alternate proton acceptor for the Schiff base. On the basis of these findings, a mechanism is proposed for proton pumping in BPR. Because the pKa of Glu142 is near the pH of its native marine environment, changes in pH may act to modulate its function in the cell.

The NOP-1 gene from the eukaryote Neurospora crassa, a filamentous fungus, has recently been shown to encode an archaeal rhodopsin-like protein NOP-1. To explore the functional mechanism of NOP-1 and its possible similarities to archaeal and visual rhodopsins, static and time-resolved Fourier transform infrared difference spectra were measured from wild-type NOP-1 and from a mutant containing an Asp-->Glu substitution in the Schiff base (SB) counterion, Asp131 (D131E). Several conclusions could be drawn about the molecular mechanism of NOP-1: (1) the NOP-1 retinylidene chromophore undergoes an all-trans to 13-cis isomerization, which is typical of archaeal rhodopsins, and closely resembles structural changes of the chromophore in sensory rhodopsin II; (2) the NOP-1 SB counterion, Asp131, has a very similar environment and behavior compared with the SB counterions in bacteriorhodopsin (BR) and sensory rhodopsin II; (3) the O-H stretching of a structurally active water molecule(s) in NOP-1 is similar to water detected in BR and is most likely located near the SB and SB counterion in these proteins; and (4) one or more cysteine residues undergo structural changes during the NOP-1 photocycle. Overall, these results indicate that many features of the active sites of the archaeal rhodopsins are conserved in NOP-1, despite its eukaryotic origin.

Background & Aims Using high-density human recombinant protein microarrays, we identified two potential biomarkers, kelch-like 12 (KLHL12) and hexokinase-1 (HK1), in primary biliary cirrhosis (PBC). The objective of this study was to determine the diagnostic value of anti-KLHL12/HK1 autoantibodies in PBC. Initial discovery used sera from 22 patients with PBC and 62 non-PBC controls. KLHL12 and HK1 proteins were then analysed for immunoglobulin reactivity by immunoblot and enzyme-linked immunosorbent assay (ELISA) in two independent cohorts of PBC and disease/healthy control patients. Methods Serum samples from 100 patients with PBC and 165 non-PBC disease controls were analysed by immunoblot and samples from 366 patients with PBC, 174 disease controls, and 80 healthy donors were tested by ELISA. Results Anti-KLHL12 and anti-HK1 antibodies were each detected more frequently in PBC compared with non-PBC disease controls (P

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

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.

We present a synchrotron x-ray diffraction study of melting in stacks of two-dimensional crystalline arrays of the membrane protein bacteriorhodopsin. Two distinct regimes have been found as a function of the intermembrane distance d. In the “coupled” regime for d , 250 A the temperature sTmd of the melting transition decreases with increasing d, demonstrating the effect of the repulsive membrane interactions on the intramembrane protein ordering. For d . 250 A a “decoupled” regime is found with higher T p m independent of d. Below T p m a solid-liquid-solid reentrant behavior is observed as d is increased. [S0031-9007(99)08795-5]

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho --MII difference spectra were obtained at 10 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates alpha-helix movement.

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.

Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

Bacteriorhodopsin is a light-driven proton pump, which undergoes a photocycle consisting of several distinct intermediates. Previous studies have established that the M →N step of this photocycle involves a major conformational change of membrane embedded α-helices. In order to further investigate this conformational change, we have studied the photocycle of the high pH form of the mutant Asp-85 →Asn (D85Nalk). In contrast to wild type bacteriorhodopsin, D85Nalk has a deprotonated Schiff base and a blue-shifted absorption near 410 nm, yet it still transports protons in the same direction as wild type bacteriorhodopsin (Tittor, J., Schweiger, U., Oesterhelt, D. and Bamberg, E.(1994) Biophys. J., 67, 1682-1690). Resonance Raman spectroscopy of D85Nalk and D85Nalk regenerated with retinal labeled at the C-15 position with deuterium reveals the existence of an all-trans configuration of the chromophore. Fourier transform infrared difference spectroscopy shows that the photocycle of this light-adapted form involves similar events as the wild type bacteriorhodopsin photocycle including the M →N protein conformational change. These results help to explain the ability of D85Nalk to transport protons and demonstrate that the M →N conformational change can occur even in the photocycle of an unprotonated Schiff base form of bacteriorhodopsin.

While the strong biotin-avidin interaction has been widely used for the detection of biomolecules, its irreversibility complicates their isolation. We report the synthesis of a photocleavable biotin derivative (PCB) which eliminates many limitations of existing methods. This reagent contains a biotin moiety linked through a spacer arm to a photocleavable moiety, which reacts selectively with primary amino groups on any substrate. In experiments using [leucine]-enkephalin as a model substrate, we show that PCB retains its high affinity toward avidin/streptavidin and allows rapid (< 5 min) and efficient (> 99%) photorelease of the substrate in a completely unaltered form. Photocleavable biotins should be useful in numerous applications involving the isolation of proteins, nucleic acids, lipids, and cells.

Bovine rod rhodopsin and membrane-carboxyl group mutants are expressed using the recombinant baculovirus expression system. Biosynthesis of wild-type and the mutant D83N is normal. The mutations E122L and E134D/R affect glycosylation and translocation. After regeneration, purification and reconstitution in retina lipids a wild-type photosensitive pigment with spectral and photolytic properties identical to native bovine rod rhodopsin is generated. Only the mutations D83N and E122L affect the spectral properties and then only slightly. All mutations induce a shift in the Meta I ↔ Meta II equilibrium towards Meta I (E134D/R) or Meta II (D83N, E122L). FT-IR analysis shows that the mutation E134D/R does not significantly affect the carboxyl-vibration region but, in particular in the case of E134R, affects secondary structural changes upon Meta II formation. E122L also has an effect on secondary structural changes and in addition eliminates a negative band at 1728 cm−1. The mutation D83N removes a pair of negative/positive bands from the carboxyl-vibration region, indicating that Asp83 stays protonated upon formation of Meta II but undergoes a change in hydrogen bonding.

Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.

FTIR difference spectroscopy has been used to study the role of cysteine residues in the photoactivation of rhodopsin. A positive band near 2550 cm-1 with a low frequency shoulder is detected during rhodopsin photobleaching, which is assigned on the basis of its frequency and isotope shift to the S-H stretching mode of one or more cysteine residues. Time-resolved studies at low temperature show that the intensity of this band correlates with the formation and decay kinetics of the Meta II intermediate. Modification of rhodopsin with the reagent NEM, which selectively reacts with the SH groups of Cys-140 and Cys-316 on the cytoplasmic surface of rhodopsin, has no effect on the appearance of this band. Four other cysteine residues are also unlikely to contribute to this band because they are either thio-palmitylated (Cys-322 and Cys-323) or form a disulfide bond (Cys-110 and Cys-187). On this basis, it is likely that at least one of the four remaining cysteine residues in rhodopsin is structurally active during rhodopsin photoactivation. The possibility is also considered that this band arises from a transient cleavage of the disulfide bond between cysteine residues 110 and 187.

Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorhodopsin (BR), a light-driven proton pump, share residues which have been identified as critical for BR functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions both as the Schiff base counterion and proton acceptor. We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption unlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths. The mutated proteins D76N and D76A are also fully functional as phototaxis receptors in contrast to BR, where the analogous substitutions block proton transport. D76N was also found to exhibit a spectrally normal SR587-->S373 transition. However, FTIR difference spectroscopy reveals that two bands in the SR587-->S373 difference spectrum at 1766/1749 cm-1 (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, although no changes are observed in the carboxylate region. In addition, the kinetics and yield of this photoreaction are altered. On this basis, it is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is protonated in SR-I and undergoes an increase in its hydrogen bonding during the SR587-->S373 transition. This model accounts for the difference in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype. Interestingly, parallels exist between this residue and Asp83 in the visual receptor rhodopsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodopsin activation.

FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.

Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis have been used to investigate structural changes which occur during rhodopsin photoactivation at the level of individual amino acid residues. The rhodopsin-->bathorhodopsin FTIR difference spectra of the mutants Asp-83-->Asn (D83N) and Glu-134-->Asp (E134D) incorporated into membranes are similar to that of native rhodopsin in the photoreceptor membrane, demonstrating that the retinal chromophores of these mutants undergo a normal 11-cis to all-trans photoisomerization. Two bands assigned to the C = O stretching mode of Asp and/or Glu carboxylic acid groups are absent in the D83N rhodopsin-->metarhodopsin II FTIR difference spectrum. Corresponding changes are not observed in the carboxylate C = O stretching region. The most straightforward explanation is that the carboxylic acid group of Asp-83 remains protonated in rhodopsin and its bleaching intermediates but undergoes an increase in its hydrogen bonding during the metarhodopsin I-->metarhodopsin II transition. The mutant E134D produced a normal rhodopsin-->bathorhodopsin and rhodopsin-->metarhodopsin II difference spectrum, but a fraction of misfolded protein was observed, supporting earlier evidence that Glu-134 plays a role in proper protein insertion and/or folding in the membrane.

Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-->Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adapted Y185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C=N syn-->all-trans isomerization of the retinal chromophore which produces a species similar to bR570 and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bR570 and O-like species and resulted in a decreased all-trans:13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shown by stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated in Y185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.

The light-dark adaptation, photocycle kinetics, and acid-induced blue formation of the bacteriorhodopsin (bR) mutant Tyr-185-->Phe (Y185F) expressed in Halobacterium halobium have been investigated by both static and time-resolved visible absorption spectroscopy. Evidence is presented that a pH-dependent equilibrium exists between a bR570-like form (bRY185F570) and a red-shifted species in the light-adapted form of Y185F. In two related papers, we show that this species has vibrational features similar to the O intermediate. Key findings are that light adaptation causes formation of a purple species similar to bR570 and a second long-lived red-shifted species with a lambda max near 630 nm, well above the pH for the acid-induced blue transition. The concentration of the red-shifted species is pH- and salt-dependent, decreasing reversibly at high pH and high ionic strength. The dark-adapted state of Y185F also contains a small amount of the red-shifted species which is reversibly titratable. Dark adaptation is much slower than wild-type bR and causes a parallel decay of light-adapted bR and the red-shifted species. Time-resolved visible absorption spectroscopy reveals that the purple and the red-shifted species undergo separate photocycles. The purple species exhibits a relatively normal photocycle except for an increased rate of M formation kinetics. The red-shifted species has a photocycle involving a red-shifted K intermediate and a second longer lived intermediate possibly similar to N. The apparent absence of an O intermediate in the late photocycle of Y185F is attributed to cancellation by depletion bands due to the photoreacting red-shifted species.(ABSTRACT TRUNCATED AT 250 WORDS)

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousche et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.

Invertebrate rhodopsin is the primary photoreceptor found in the eyes of cephalopods. Importantly, unlike vertebrate rhodopsin, invertebrate rhodopsins such as sepia rhodopsin (s-Rh) can be activated by light and then rapidly cycled back to the original state with a second red-shifted photon, thereby facilitating a variety of novel biophysical studies. Additionally, invertebrate rhodopsins can bind to the ubiquitous β-Arrestin2 which is used in regulating signal transduction in many GCPRs. In this study, we used static and time-resolved FTIR difference spectroscopy to investigate the photocycle of s-Rh complexed to β-Arrestin2. In the spectrum of s-Rh alone, difference spectra obtained using two colors to cycle between the ground (rho) and acid meta state show an 11-cis to all-trans photocycle as previously described. Several bands between 1750-1700 cm-1 are assigned using a D2O induced shift to an as yet unknown carboxyl groups. Other large bands are seen especially in the amide I and II regions which indicate significant backbone structural changes. Upon addition of β-Arrestin2, the difference spectrum is altered, especially in the amide I and II regions, reflecting additional structural changes occurring in β-Arrestin2 upon photoactivation. A negative band at 1742 cm-1 was shifted higher in the complex indicating the β-Arrestin2 is perturbing at least one carboxyl group in s-Rh. In order to assign these changes, total 15N isotope labeling of β-Arrestin2 was utilized. Comparison of difference spectra from s-Rh complexes containing unlabelled and 15N labeled β-Arrestin2 reveals ∼ 3 cm-1 downshift of a negative/positive feature at 1668/1655 cm-1 indicating that these bands reflect at least partially conformational changes of the β -arrestin involving α-helical structure. These results offer a promising new tool to investigate the molecular mechanism of β-Arrestin interactions with GCPRs to regulate downstream signaling.

The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96—-Ala and Asp-96—-Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96—-Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L—-M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N—-O or O—-bR transition.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25–1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.

Proteorhodopsins are an extensive family of photoactive membrane proteins found in proteobacteria distributed throughout the world's oceans which are often classified as green- or blue-absorbing (GPR and BPR, respectively) on the basis of their visible absorption maxima. GPR and BPR have significantly different properties including photocycle lifetimes and wavelength dependence on pH. Previous studies revealed that these different properties are correlated with a single residue, Leu105 in GPR and Gln105 in BPR, although the molecular basis for the different properties of GPR and BPR has not yet been elucidated. We have studied the unexcited states of GPR and BPR using resonance Raman spectroscopy which enhances almost exclusively chromophore vibrations. We find that both spectra are remarkably similar, indicating that the retinylidene structure of GPR and BPR are almost identical. However, the frequency of a band assigned to the retinal C13-methyl-rock vibration is shifted from 1006 cm (-1) in GPR to 1012 cm (-1) in BPR. A similar shift is observed in the GPR mutant L105Q indicating Leu and Gln residues interact differently with the retinal C13-methyl group. The environment of the Schiff base of GPR and BPR differ as indicated by differences in the H/D induced down-shift of the Schiff base vibration. Residues located in transmembrane helices (D-G) do not contribute to the observed differences in the protein-chromophore interaction between BPR and GPR based on the Raman spectra of chimeras. These results support a model whereby the substitution of the hydrophilic Gln105 in BPR with the smaller hydrophobic Leu105 in GPR directly alters the environment of both the retinal C13 group and the Schiff base.

The secondary structure of bacteriorhodopsin has been investigated by polarized Fourier transform infrared spectroscopy combined with hydrogen/deuterium exchange, isotope labeling and resolution enhancement methods. Oriented films of purple membrane were measured at low temperature after exposure to H2O or D2O. Resolution enhancement techniques and isotopic labeling of the Schiff base were used to assign peaks in the amide I region of the spectrum. alpha-helical structure, which exhibits strong infrared dichroism, undergoes little H/D exchange, even after 48 h of D2O exposure. In contrast, non-alpha-helical structure, which exhibits little dichroism, undergoes rapid H/D exchange. A band at 1,640 cm-1, which has previously been assigned to beta-sheet structure, is found to be due in part to the C = N stretching vibration of protonated Schiff base of the retinylidene chromophore. We conclude that the membrane spanning regions of bR consist predominantly of alpha-helical structure whereas most beta-type structure is located in surface regions directly accessible to water.

The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.

Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue bymore » glycine preserves the normal protein structural changes during the photocycle.« less

Data in the literature suggest a finely tuned interaction between ligand (11-cis-retinal) and protein (opsin) in order to allow very efficient photoactivation of the ligand and highly vectorial rhodopsin activation with a huge increase in receptor activity. We have further investigated this interaction using ligand homologues, 13C-ligand labelling or 15N-protein labelling, in combination with Fourier transform infrared (FT-IR) and solid-state magic angle spinning (ss-MAS)-NMR spectroscopy. Using 1D rotational resonance (RR) or double-quantum heteronuclear local field (2Q-HLF) ss-MAS-NMR we report the first structure refinement of the rhodopsin chromophore in situ. These measurements yield a specification of the torsional strain in the for isomerization essential C10-C13 segment of the chromophore. This strain is thought to contribute to the high rate and stereospecificity of the photoisomerization reaction. In agreement with previous data, the C10-C13 segment region reaches a relaxed all-trans configuration at the lumirhodopsin photointermediate. MAS-NMR analysis of [15N]lysine-labelled rhodopsin reveals the presence of a 'soft' counterion, requiring intermediate water molecules for stabilization. FT-IR studies on [2H]tyrosine-labelled rhodopsin demonstrate participation of several tyrosin(at)e residues in receptor activation. One of these, probably Tyr268, is already active at the bathorhodopsin stage. Finally, the effect of ligands with single additional methyl substituents in the C10-C12 region has been investigated. They do not affect the general activation pathway, but perturb the activation kinetics of rhodopsin, suggesting steric interference with protein residues. Possible implications of these results for a structural role of water residues will be discussed, as well.

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

Channelrhodopsins (ChRs) from green algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs in order to bioengineer their properties for specific optogenetic applications. Most studies have focused so far on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Here, low-temperature and time-resolved rapid scan FTIR-difference spectroscopy, along with kinetic UV-Visible transient absorbance spectroscopy, are applied to study protein conformational changes occurring during the photocycle of the red-shifted channelrhodopsin-1 from Chlamydomonas augustae (CaChR1). Substitutions with isotope-labeled retinals or the retinal analog A2, site-directed mutagenesis, hydrogen-deuterium exchange and H218O substitution are used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to CrChR2, almost exclusively all-trans to 13-cis isomerization of the retinal chromophore, similar to the primary phototransition of bacteriorhodopsin (BR). A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1 which, along with amide I bands, reflects alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2 indicating a more heterogeneous environment. Based on the data from mutations involving the residues Glu169 and Asp299 (homologs of the Asp85 and Asp212 Schiff base counterions in BR), as well as Phe139 (Lys132 in the C1C2 chimera located within hydrogen bonding distance of the homologs of Asp85 and Asp212), we propose a model for protonation changes occurring near the Schiff base during the P1 and P2 steps of the photocycle.

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900cm−1 due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.

TWO-DIMENSIONAL assemblies of membrane proteins (see ref. 1, for example) such as bacteriorhodopsin are of current interest because of their potential application in technological areas as diverse as molecular electronics and optical switching2, molecular sieves3,4 and the lithographic fabrication of nanometre-scale patterns5,6. Here we report that bacteriorhodopsin7–9 can retain its folded native structure to temperatures as high as 140 °C when incorporated in multilayer structures of self-assembled, ordered films. Synchrotron X-ray scattering reveals that, under hydrated conditions, the two-dimensional lattice in multilayer films exhibits a reversible solid–liquid transition at about 69 °C, followed by irreversible denaturing of the bacteriorhodopsin at about 90 °C. But in dry films the melting transition and denaturation are suppressed up to 140 °C. These results suggest that it may be feasible to use multilayer assemblies of functional proteins and enzymes10,11 in high-temperature applications.

Channelrhodopsins (ChRs) control phototaxis in green algae and function as light-gated cation channels when expressed in animal cells. Because ChRs can be functionally expressed in neuronal membranes, this distinct family of microbial rhodopsins have rapidly become an important tool in neuroscience. While the light-activated molecular changes occurring in channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) have been extensively studied, little is known about such changes in the diverse groups of other ChRs including the major class of channelrhodopsin-1 (ChR1). Here, we have characterized the structure and molecular changes in ChR1 from Chlamydomonas augustae (CaChR1). This ChR has properties advantageous for light modulated neuronal control including a red-shifted λmax and slow light inactivation compared to CrChR2 (Hou, S. et al. (2012) Photochem Photobiol 88, 119-128). Near-infrared confocal resonance Raman spectroscopy (RRS) reveals that in contrast to CrChR2, which contains a mixture of retinal isomers, the retinal chromophore structure of CaChR1 is almost completely all-trans in the light-adapted state similar to many microbial rhodopsins including bacteriorhodopsin. In addition, unlike other ChR1s, such as CrChR1 (ChR1 from Chlamydomonas reinhardtii), which exhibit significant shifts in λmax at different pHs, the RRS of CaChR1 including the ethylenic frequency which reflects λmax remains largely unaltered over a wide pH range. This insensitivity to pH is despite the presence of the residue Glu87 (CrChR1 sequence numbering) which has been previously associated with pH sensitivity. Low-temperature FITR difference-spectroscopy reveals that the primary phototransition of CaChR1 to the K photointermediate involves an all-trans to 13-cis retinal isomerization and significant changes in the protein structure including structural changes in the peptide backbone, Asp/Glu residues, Cys residues and internal water molecules. These results are discussed in terms of possible differences in the molecular mechanism of various ChRs.