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The RHO gene encodes the G‐protein‐coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light‐activated state by solution and MAS‐NMR spectroscopy. We found two long‐lived dark states for the G90D mutant with the 11‐cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11‐cis retinal caused by the mutation‐induced distortion on the salt bridge formation in the binding pocket. Time‐resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle., Rhodopsin is the major dim light receptor in vertebrate eyes. Numerous mutations associated with impaired visual cycles are known. The G90D mutation leads to a constitutively active mutant form of rhodopsin that causes congenital stationary night blindness (CSNB). We investigated the consequences of this mutation on the visual cycle, both in terms of structural aspects and dynamic changes.

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.

To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected "GxyG" host-guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest-neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above-the-average preference for turn-like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above-the-average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of (3) J(H(N) ,H(α) ) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPII↔β equilibrium indicating a significant residue dependent temperature dependence of the peptides' conformational ensembles. These results suggest that nearest-neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy-entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/β dominated ensembles at physiological temperatures.