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

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. 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 are usually highly complex. Linking the spectral response to specific molecular events is a major obstacle. Here, we investigate a cyanobacteriochrome 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 regimes. 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.