Nanosecond protein dynamics in a red/green Cyanobacteriochrome revealed by transient IR spectroscopy

TL;DR

This study uses transient IR spectroscopy to reveal nanosecond protein dynamics in cyanobacteriochrome photoreceptors, identifying ground state intermediates and structural changes, advancing understanding of their functional mechanisms.

Contribution

It combines visible and IR transient absorption spectroscopy with advanced analysis methods to elucidate early protein dynamics and intermediates in cyanobacteriochrome photoreceptors.

Findings

Identified two ground state intermediates for Pr*

Discovered four intermediates for Pg*

Reported structural change in protein backbone

Abstract

Over the last decades, photoreceptive proteins were extensively studied with biophysical methods to gain a fundamental understanding of their working mechanisms and further guide the development of optogenetic tools from them. Time-resolved infrared (IR) spectroscopy is one of the key methods to access their functional non-equilibrium processes with high temporal resolution, but has the major drawback that experimental data is usually highly complex. Linking the spectral response to specific molecular events is a major obstacle. Here, we investigate a cyanobacteriochrome (CBCR) photoreceptor with a combined approach of transient absorption spectroscopy in the Visible and IR spectral regions. We obtain kinetic information in both spectral regions by analysis with two different fitting methods, global multiexponential fitting and lifetime analysis. We investigate the ground state dynamics that follow photoexcitation in both directions of the bi-stable photocycle (Pr* and Pg*) in the nanosecond and microsecond time regime. We find two ground state intermediates associated with the decay of Pr* and four with Pg* and report the macroscopic time constants of their interconversions. One of these processes is assigned to a structural change in the protein backbone. At later delay times, the spectroscopic responses are stretched out in time, an effect that can be better described by the lifetime analysis than the global fit. We ascribe it to the intrinsic ruggedness of the free energy landscape of proteins.