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].

Analogues of E. coli dihydrofolate reductase (DHFR) containing modified amino acids at single, predetermined sites have been prepared. This was accomplished by the use of the DHFR gene containing an engineered nonsense codon (TAG) at the positions corresponding to Val-10 and Asp-27. Misacylated suppressor tRNAs activated with the modified amino acids of interest were employed for the suppression of the nonsense codons in a cell free protein biosynthesizing system, thereby permitting the elaboration of the desired protein analogues. In this fashion, the aspartic acid analogues erythro-carboxyproline, cysteic acid, β,β-dimethylaspartic acid, α-methylaspartic acid, erythro- and threo-β-methylaspartic acid, N-methylaspartic acid, and phosphonoalanine were incorporated into one or both of the aformentioned positions. Although a number of these analogues were incorporated only in low yield, a modification of the strategy has suggested how this might be improved significantly. The derived proteins were purified and then characterized by their mobility on polyacrylamide gels in comparison with wild-type DHFR. Representative DHFRs modified at position 10 were also degraded by defined proteolysis with Glu-C endoproteinase; the fragments containing the modified amino acids were shown to have the same chromatographic properties on reverse phase HPLC as authentic synthetic standards. Individual analogues were assayed for their abilities to bind to the substrate analogue methotrexate and to convert dihydrofolate to tetrahydrofolate. DHFR analogues containing erythro- and threo-β-methylaspartic acid and β,β-dimethylaspartic acid were all shown to mediate tetrahydrofolate production 74−86% as efficiently as wild-type DHFR under conditions of multiple substrate turnover. Analysis of the rates of tetrahydrofolate production in the presence of NADPH and NADPD at two pH values suggests that this was due to rate-limiting hydride transfer from NADPH bound to DHFR analogues whose active site had been altered structurally.

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Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT) was replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains producing specific amber suppressor tRNA's to express luciferase with different substitutions at this position. The incorporation of Leu, Lys, Tyr, or Gln at this position reduced the thermostability of mutated luciferases. The color of emitted light changed upon substitution from yellow-green (λmax 582 nm) for the wild-type enzyme having Ser286 to, for example, red (λmax 622 nm) for luciferase having Leu286. For further evaluation of the structural relationship between the amino acid position at 286 and the wavelength of emitted light, we used the method of in vitro incorporation of unnatural amino acids, which involves readthrough of a nonsense (UAG) codon by a misacylated suppressor tRNA. The amino acids incorporated at position 286 in this fashion included O-glucosylated serine, serine phosphonate, tyrosine phosphate, and tyrosine methylenephosphonate. The wavelength of light emitted by the luciferase analogues was measured. While the introduction of serine phosphonate and glucosylated serine did not change the λmax of light produced by luciferase, the incorporation of tyrosine phosphate and tyrosine methylenephosphonate into position 286 altered the spectra of emitted light compared with those of Ser286 and Tyr286. The pH dependence of the wavelength of light emitted by the luciferases containing the negatively charged phosphorylated Tyr analogues was demonstrated and could be rationalized in terms of the pK a's of the phosph(on)ate oxygens.