Temperature Dependence of the Krokinobacter rhodopsin 2 Kinetics

ABSTRACTHere we applied target analysis to a temperature dependent flash photolysis dataset of the light-driven sodium ion pump Krokinobacter rhodopsin 2 (KR2) at sodium pumping conditions. With an increase in temperature from 10 – 40 °C, the overall photocycle duration was accelerated by a factor of six, while single transitions like the L to M transition increased by a factor of 40. Using kinetic modeling with the Eyring constraint as well as spectral corrections on the datasets the spectral position as well as the equilibria of the different photointermediates could be resolved. The results provide further insight into KR2s photocycle and energetics.STATEMENT OF SIGNIFICANCEKR2 is the most prominent member of the new class of non-proton cation pumps, as it represents an interesting new optogenetic tool. Despite widespread biophysical investigations, the molecular mechanisms of light-induced sodium pumping in KR2 are still not sufficiently understood. Therefore, an expanded set of thermodynamic parameters is essential for a complete picture. Our study of the KR2 photocycle shows that different steps in the photocycle are affected differently by temperature changes. Rigorous data analysis provides strong evidence that the transient states observed in time-resolved experiments represent rather equilibria between the different photocycle intermediates than pure intermediates. Gaining access to the dynamics and energetics of KR2 helps to answer long standing open questions concerning the molecular mechanism of cation pumping.

Structure and mechanisms of sodium-pumping KR2 rhodopsin

Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 fromKrokinobacter eikastuswas discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

Protein conformational alterations induced by the retinal excited state in proton and sodium pumping rhodopsins

Light-induced charge redistribution in the retinal chromophore, followed by a protein conformational change.