Long-term tracking of protein conformational dynamics through single-molecule thermal snapshot spectroscopy

• Applicant: Dr. Christopher Engelhard
• Subject Area: Biophysics
• Term: from 2018 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 399893536

Protein structures from X-ray crystallography are detailed but frozen images: they lack information about how proteins fluctuate, adapt and bind to other biomolecules, and which movements are involved in performing their functions. Similarly, standard single-molecule spectroscopy is well suited to studying the behaviour of individual proteins at room temperature, but is inherently limited, both in its time resolution and in maximum observation times. This application proposes a general method to access protein dynamics at the single-molecule level. The central idea is to freeze-quench a single biomolecule repeatedly at different stages of its natural motion, providing a stop-motion movie of its dynamics. This will be achieved through fast (microsecond) freezing and thawing cycles, between a low temperature where motion is frozen and a high temperature where motion proceeds naturally. This method holds all the advantages of single-molecule observations: it eliminates ensemble averaging and does not require any synchronization, for each single molecule is in a single state at any time. Whereas other techniques, e.g., cryo-electron microscopy, do reveal the spread of conformations found in ensembles of single molecules, this method will record the sequence of conformations, providing unique insight into pathways in the potential energy landscape.Initial experiments in M. Orrit's lab have demonstrated the feasibility of microsecond temperature cycles by means of laser heating in a cold environment. This proposal sets two objectives to turn these exploratory experiments into a reliable experimental method:a) to apply the temperature cycle method to biomolecules whose dynamics is well characterized, and thereby to provide proof of the reliability of the method;b) to apply the technique to open problems in protein dynamics on metallo-proteins and adenylate kinase, in collaboration with world-class groups in the field of protein dynamics research.

• DFG Programme: Research Fellowships
• International Connection: Netherlands
• Host: Professor Dr. Michel Orrit