His75−Asp97 Cluster in Green Proteorhodopsin

Abstract

The proteorhodopsin (PR) family found in bacteria near the ocean's surface consists of hundreds of PR variants color-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using (13)C and (15)N MAS NMR, we were able to prove for green PR that His75 forms a pH-dependent H-bond with the primary proton acceptor Asp97, which explains its unusually high pK(a). The functional role of His75 has been studied using site-directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PR(H75N) showed that the primary reaction dynamics is retained, while flash photolysis experiments revealed an accelerated photocycle. Our data show the formation of a pH-dependent His-Asp cluster which might be typical for eubacterial retinal proteins. Despite its stabilizing function, His75 was found to slow the photocycle in wild-type PR. This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.

Unexpected effects of third-order cross-terms in heteronuclear spin systems under simultaneous radio-frequency irradiation and magic-angle spinning NMR

“…3, which compares the predicted splitting in the 13 C spectrum under LG decoupling from Eq. (31) with numerical simulations of both LG and FSLG decoupling under 10 kHz MAS (see Sec.…”

“…Figure 1(c) shows the overlay of two 13 C, 1 H HETCOR spectra of terbutaline sulfate (TBS) form B, obtained at 500 MHz 1 H NMR frequency using cross-polarisation mixing times of 150 and 350 μs under otherwise identical conditions (full experimental details can be found in Ref. 25).…”

“…The long-range cross peaks (black) involving the two C6 resonances have subtly different apparent 1 H shifts compared to short-range correlations involving the same hydrogens (red). The short-range one-bond CH correlation peaks at the two times are directly superimposable (the peaks from the long contact time experiment are greyed out for clarity Related effects may be observed for dilute spins, such as 13 C, when homonuclear decoupling is applied to the 1 H spins. Figure 2 shows the results (solid lines) of spin-echo experiments used to measure the 1 J CH couplings for the CH and CH 3 resonances of L-alanine.…”

“…However, it is only with the development of 1 H homonuclear decoupling schemes suitable for application under moderate to fast MAS frequencies, e.g., Frequency-Switched Lee-Goldburg (FSLG), 5 Phase-Modulated Lee-Goldburg (PMLG), 6 and DUMBO, 7 coupled with advances in RF consoles, that such experiments have started to be more widely employed. 8,9 For example, 13 C, 1 H heteronuclear correlation (HETCOR) experiments at moderate to fast MAS frequencies 10,11 have been widely applied to a variety of significant material types, such as proteins, 12,13 pharmaceuticals, 14,15 silica-supported catalytic complexes, 16,17 organic-templated microporous materials, 18 inorganic-organic hybrid materials, 19 as well as chemical problems, such as the characterisation of weak hydrogen bonding in sugars 20 and the solution of structures from powder diffraction data. 21 Heteronuclear 13 C{ 1 H} spin-echo experiments employing 1 H homonuclear decoupling during the a) Author to whom correspondence should be addressed.…”

“…24 This paper investigates two experimental phenomena associated with the application of homonuclear 1 H decoupling in heteronuclear 13 C, 1 H solid-state MAS NMR; firstly, changes in the apparent 1 H chemical shift of correlation peaks in 1 H, 13 C two-dimensional experiments depending on whether there is or is not a one-bond 13 C-1 H dipolar coupling, and secondly, the observation of a much faster dephasing in a 13 C{ 1 H} heteronuclear spin-echo experiment under homonuclear 1 H decoupling as compared to heteronuclear 1 H decoupling. An analytical description is presented that, supported by numerical simulations, shows that these two phenomena have a common explanation in terms of thirdorder cross terms involving the 13 C, 1 H heteronuclear dipolar coupling.…”