Dresden 2020 – scientific programme

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BP: Fachverband Biologische Physik

BP 33: Protein Structure and Dynamics

BP 33.4: Talk

Thursday, March 19, 2020, 10:15–10:30, ZEU 250

Following the formation of PYP’s photocycle intermediates on a femtosecond to millisecond timescale with a site-specific IR label — •Larissa Blankenburg, Luuk J.G.W. van Wilderen, and Jens Bredenbeck — Goethe-Universität, Institut für Biophysik, Max-von-Laue-Str. 1, 60438 Frankfurt am Main, Germany

The photocycle dynamics of Photoactive Yellow Protein (PYP) that are induced by excitation with blue light occur on a timescale ranging from femtoseconds to seconds. Local dynamic information about the protein can be obtained by the use of the vibrational label thiocyanate (SCN) that can be inserted site-specifically at any desired position by cysteine mutation and cyanylation. The CN stretch vibration is highly sensitive to polarity and hydrogen-bonding interactions and thus allows to probe local structural changes during PYP’s photocycle.

With transient fs-ms infrared spectroscopy on SCN-labeled PYP mutants we followed most part of the photocycle from chromophore isomerization (ps) and protonation (µs) to partial unfolding of the protein (ms). The data revealed spectral changes corresponding to alterations in the local environment of the non-perturbing label, providing dynamic site-specific structural information for multiple observed photocycle intermediates. While the site resolution in infrared spectroscopy of unlabeled proteins is generally limited to a few marker bands (e.g. of the chromophore or specific side chains), vibrational labels can be inserted at almost every location improving the structural resolution and investigation of proteins and may resolve new intermediates.