Capturing Functionally Relevant Protein Motions At The Atomic Level: Femtosecond Time Resolved Serial Crystallography Of Ligand Dissociation Of Carboxy-Myoglobin

The recent advent of X-Ray free electron lasers with highest brilliance and femtosecond pulses opens new possibilities for time-resolved protein crystallography [Miller, R.J.D, Science, 2014, 343, 1108-1116]. A fundamental biophysical question becomes accessible experimentally now: The investigation of protein dynamics with all atomic resolution on the shortest biochemically relevant timescale around 100 fs. Here is where bond-breaking events occur, which in turn translate into secondary and tertiary structure changes and cause a protein to fulfill its function over a wide range of timescales. The question is what is the structure-function correlation in proteins and which time and length scales are involved. We present results on Myoglobin in complex with Carbonmonoxide as ligand. The actual bond-breaking event is expected faster than 50 fs [Armstrong, M.R. et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 4990]. Our experiment accesses the dynamics in the time window between 0 and 2 ps with 100 fs steps to resolve both the ligand dissociation and the “talking” coordinate mediated through the proximal histidine motion involved in allosteric communication of the ligation state. The heme moiety doming and the overall protein motion coupled to the bond breaking process are resolved and the data clearly demonstrate coupling to collective coordinates on exceptionally fast time scales. Experiments were performed at Linac Coherent Light Source (LCLS) under atmospheric pressure at room temperature using a solid target photo-crystallography chip for sample delivery [Mueller C. et al., Structural Dynamics, 2015, 2, 054302]. Up to 10.000 individual crystal samples per chip can be trapped and mapped using absorption spectroscopy, allowing to achieve effective 100% (u003e90%) hit rates. The technological approach allows for further experiments to atomically resolve protein dynamics.