Tuning the primary reaction of channelrhodopsin-2 by imidazole, pH, and site-specific mutations.
Femtosecond time-resolved absorption measurements were performed to investigate the influence of the pH, imidazole concentration, and point mutations on the isomerization process of Channelrhodopsin-2. Apart from the typical spectral characteristics of retinal isomerization, an additional absorption feature rises for the wild-type (wt) on a timescale from tens of ps to 1 ns within the spectral range of the photoproduct and is attributed to an equilibration between different K-intermediates. Remarkably, this absorption feature vanishes upon addition of imidazole or lowering the pH. In the latter case, the isomerization is dramatically slowed down, due to protonation of negatively charged amino acids within the retinal binding pocket, e.g., E123 and D253. Moreover, we investigated the influence of several point mutations within the retinal binding pocket E123T, E123D, C128T, and D156C. For E123T, the isomerization is retarded compared to wt and E123D, indicating that a negatively charged residue at this position functions as an effective catalyst in the isomerization process. In the case of the C128T mutant, all primary processes are slightly accelerated compared to the wt, whereas the isomerization dynamics for the D156C mutant is similar to wt after addition of imidazole.
Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
Frank
F
Scholz
Ernst
E
Bamberg
Christian
C
Bamann
Josef
J
Wachtveitl
D000042:Absorption / Q000187:drug effects; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology*; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D055668:Photochemical Processes / Q000187:drug effects; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013057:Spectrum Analysis; D013997:Time Factors
D007093:Imidazoles; D050505:Mutant Proteins; C029899:imidazole; D012243:Rhodopsin; D012172:Retinaldehyde