Color-tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light-induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground-state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.

Small-angle X-ray and small-angle neutron scattering (SAXS/SANS) provide unique structural information on biomolecules and their complexes in solution. SANS may provide multiple independent data sets by means of contrast variation experiments, that is, by measuring at different D 2 O concentrations and different perdeuteration conditions of the biomolecular complex. However, even the combined data from multiple SAXS/SANS sets is by far insufficient to define all degrees of freedom of a complex, leading to a significant risk of overfitting when refining biomolecular structures against SAXS/SANS data. Hence, to control against overfitting, the low-information SAXS/ SANS data must be complemented by accurate physical models, and, if possible, refined models should be cross-validated against independent data not used during the refinement. We present a method for refining atomic biomolecular structures against multiple sets of SAXS and SANS data using all-atom molecular dynamics simulations. Using the protein citrate synthase and the protein/RNA complex Sxl−Unr−msl2 mRNA as test cases, we demonstrate how multiple SAXS and SANS sets may be used for refinement and cross-validation, thereby excluding overfitting during refinement. For the Sxl−Unr−msl2 complex, we find that perdeuteration of the Unr domain leads to a unique, slightly compacted conformation, whereas other perdeuteration conditions lead to similar solution conformations compared to the nondeuterated state. In line with our previous method for predicting SAXS curves, SANS curves were predicted with explicit-solvent calculations, taking atomic models for both the hydration layer and the excluded solvent into account, thereby avoiding the use of solvent-related fitting parameters and solventreduced neutron scattering lengths. We expect the method to be useful for deriving and validating solution structures of biomolecules and soft-matter complexes, and for critically assessing whether multiple SAXS and SANS sets are mutually compatible.

The glutamatergic synapses are assembled and maintained by conserved planar cell polarity proteins.

Human guanylate‐binding protein 1 (hGBP1) belongs to the family of dynamin‐like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post‐translationally modified by attachment of a farnesyl group yielding farn‐hGBP1. In this study, hydrodynamic differences in farn‐hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size‐exclusion chromatography (SEC). In addition, we performed small‐angle X‐ray scattering (SAXS) experiments coupled with a SEC setup (SEC‐SAXS) to investigate structural properties of nonmodified hGBP1 and farn‐hGBP1 in solution. SEC‐SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal‐like conformation in nucleotide‐free solution, whereas unmodified hGBP1 forms a monomer–dimer equilibrium both in the inactive ground state in nucleotide‐free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn‐hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn‐hGBP1 that occur in the presence of nucleotides. Database All SEC‐SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.