Structural insights into the activation mechanism of melibiose permease by sodium binding

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H+, Na+, or Li+. In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na+ binding. Secondly, though Asp124 is not essential for Na+ binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na+-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na+ binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

Lorenz-Fonfria VA, Resler T, Krause N, et al. Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating. Proceedings Of The National Academy Of Sciences. 2013;110(14):E1273-E1281.The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P-2(390) and P-3(520)) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane a-helices with a t(1/2) = 60 mu s, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t(1/2) = 10 mu s), with the latter being reprotonated by aspartic acid 156 (t(1/2) = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P-4(480) state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae

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