Chemical Physics Seminar: Ultrafast Structural Dynamics Studies of Protein Function

Abstract:

Fundamental biophysical and chemical question become accessible by the study of structural dynamics starting on the ultrafast timescale. Several experimental approaches have evolved in the last decade to access more and more complex molecular systems with both better time and spatial resolution. In my talk I will address applications of multidimensional IR spectroscopy (2D-IR) and time-resolved serial crystallography using XFELs and synchrotrons.

Two-dimensional IR spectroscopy is a great tool to study structural dynamics in a wide range of systems. Its inherent real-time time resolution is on the femto- and picosecond timescale. For applications to study protein function, spectral congestion becomes the main limitation to use the structural information accessible. Using site-specific probes, such as unnatural amino acids (UAAs) which carry an IR reporter as functional group, this problem can be overcome and spatially resolved studies of protein dynamics become possible. First applications of the combined approached (2D-IR plus UAAs) included the probing of the local microenvironment in proteins with an azide or thiocyanate moiety [1] and the investigation of ultrafast conformational dynamics on the picosecond time scale by spectral diffusion. Further site-specific labels are useful for the study of vibrational energy flow processes (VET) [2,3], which are discussed to be relevant for allosteric communication in protein domains.

In contrast, the use of XFELs in time-resolved serial femtosecond crystallography (TR-SFX) experiments promises a fully resolved view into protein dynamics, both with all atomic resolution and femtosecond time-resolution. We have developed a fixed-target based serial crystallography approach and studied the ligand-dissociation of CO in Carboxy-Myoglobin by TR-SFX in experiments at LCLS and SACLA [4,5]. These experiments were carried out under low excitation conditions to access biologically relevant motions and prevent artefacts induced by multi-photon processes. While most experimental challenges were solved, including but not limited to sample delivery and timing protocol, the overall signal is extremely weak. Building on these technological developments, we have recently carried out time-resolved serial crystallography at Petra III with a time-resolution of milliseconds to seconds. Using our “Hit-and-Return” approach [6] it is possible to measure sufficient data for several time points within a single beamtime. Results show unprecedented insights into the catalytic cycle of the enzyme defluorinase and demonstrate the potential of this technology for further insights into protein function.

References

[1] L. J. G. W. van Wilderen, D. Kern-Michler, H. M. Müller-Werkmeister, J. Bredenbeck, Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy, Phys. Chem. Chem. Phys., 16, 19643-19653, 2014

[2] H. M. Müller-Werkmeister, Y.-L. Li, E.-L. W. Lerch, D. Bigourd, J. Bredenbeck, Ultrafast Hopping from Band to Band: Assigning Infrared spectra based on vibrational energy transfer, Angew. Chem. Int. Ed., 52 (24), 6214–6217, 2013

[3] H. M. Müller-Werkmeister, J. Bredenbeck, A donor-acceptor pair for the real-time study of energy flow in proteins, Phys. Chem. Chem. Phys., 16, 3261-3266, 2014

[4] S. Oghbaey*, A. Sarracini*, H. M. Ginn*, O. Pare-Labrosse, A. Kuo, A. Marx, S. W. Epp, D. A. Sherrell, B. T. Eger, Y. Zhong, R. Loch, V. Mariani, R. Alonso-Mori, S. Nelson, H. T. Lemke, R. L. Owen, A. R. Pearson, D. I. Stuart, O. P. Ernst, H. M. Müller-Werkmeister, R. J. D. Miller, Fixed Target combined with Spectral Mapping: Approaching 100% Hit Rates for Serial Crystallography, *contributed equally, Acta Cryst. D, D72, 944-955, 2016

[5] R. L. Owen, D. Axford, D. A. Sherrell, A. Kuo, O. P. Ernst, E. C. Schulz, R. J. D. Miller, H. M. Müller-Werkmeister, Low dose fixed target serial synchrotron crystallography, Acta Cryst. D, D73, 373-378, 2017

[6] E.C. Schulz*, P. Mehrabi* , H. M. Müller-Werkmeister*, F. Tellkamp , A. Jha , W. Stuart , E. Persch , R. De Gasparo , F. Diederich , E. F. Pai, R. J. D. Miller*, The hit-and-return system enables efficient time-resolved serial synchrotron crystallography, Nat. Meth., 2018 *contributed equally