Welcome to a seminar given by Dr. Eike C. Schulz from Max-Planck Institute for Structural Dynamics and Matter in Hamburg, Germany. He will present recent developments investigation structural dynamics of proteins using time resolved serial synchrotron crystallography.
Venue: MAX IV Laboratory, 4th floor MAX II room
Time: Tuesday, June 18 2019, 14:00 – 15:00
Title: Watching enzymes at work with Time-resolved Serial Synchrotron Crystallography
The functional characterization of biomolecular function and catalysis requires not only an understanding of the three-dimensional structural ensemble but also a correlation with its time-dependent changes. Time-resolved serial synchrotron crystallography (TR-SSX) simplifies accessing both structure and dynamics at the prevalent biological time-scales (> ns). To streamline data collection we have applied the recently developed the hit-and return (HARE) approach, which allows to collect several time points along the reaction coordinate of an enzyme during a single synchrotron beamtime. This contrasts conventional methods, which require several hours up to several beamtimes per time-point. We used the HARE approach in combination with a classic optical pump-probe setup to capture 18 time points from 30 milliseconds to 30 seconds during the non-reversible turnover cycle of fluoroacetate dehalogenase (FAcD). These time points include all key states involved in enzymatic C-F bond cleavage: substrate binding and reorientation, covalent-intermediate formation, location of the water nucleophile and product release. In total four substrate turnovers can be observed between the two subunits, which are highly coupled but display different conformations. Reactivity is coupled to molecular breathing, expressed by dynamic changes in lateral FAcD dimensions and modulation in water content pointing at an allosteric communication pathwaybetween the two subunits. These results demonstrate the excellent suitability of TR-SSX to unravel biomolecular catalysis and provide key insights into protein dynamics.To circumvent the use of physical or chemical triggers for reaction initiation, such as temperature-, pH jumps, or light pulses for systems that are not naturally amenable to light activation we developed an in-situ mixing setup. To this end we extended the HARE approach with a piezo droplet injector. Our “Liquid Application Method for time-resolved Analysis”(LAMA) in combination with the HARE approach uses minimal amounts of protein (1-3 mg) and ligand (1.5-3 µl) per time point, making this scheme tremendously material efficient. Using Glucose Isomerase as a model system we were able to observe an open ring conformation of a glucose molecule in a reaction that spans several minutes in the enzyme. These newly developed methods are highly flexible, can be easily adapted to synchrotron and XFEL beamlines and are amenable to the majority of enzymatic systems, which now become accessible to time-resolved studies.