The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

Abstract

The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced (13)C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

“…This technique requires temperatures around 100 K that are also compatible with trapping of photointermediates as outlined below. DNP-enhanced MAS NMR is not yet a routine method but is applied increasingly to complex, mechanistic studies on retinal proteins (45)(46)(47)(48) and other membrane proteins (49)(50)(51).…”

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

“…We therefore selected the BC loop because no distinct functional role was known so far, and only a small ␤ turn in contrast to the full ␤ sheet, as in bacteriorhodopsin, has been observed (18,19,30,31). Other regions, such as the EF loop, are known to affect color and protein stability (22,32), none of which has been reported for the BC loop. For an effective splicing reaction, the amino acid sequence of the protein segments flanking the inteins has been modified according to the native extein sequence, as described previously (9).…”

“…Retinal was added directly to the E. coli cultures for incorporation into proteo-opsin. For DNP enhancement, the reconstituted samples were incubated with 10% H 2 O, 30% d 8 -glycerol, 60% D 2 O, and 20 mM TOTAPOL (27) overnight at 4°C as described before (22). The solution was removed completely before the sample was packed into a 3.2-mm ZrO 2 rotor.…”

“…Preparation of 14, C All-trans-retinal Proteorhodopsin Samples for DNP-enhanced NMR-All-trans-retinal 13 C-labeled at positions 14 and 15 was synthesized as described previously and was provided by Prof. Richard Brown (University of Southampton) (22). Retinal was added directly to the E. coli cultures for incorporation into proteo-opsin.…”

“…Expression, purification, and reconstitution into liposomes of pR WT were done as described previously (22). In addition, the following mutations were introduced into the pR WT WT .…”