Szabolcs Bódizs: Observing protein structural dynamics by time-resolved cryo-electron microscopy

Abstract

Proteins are dynamic systems, undergoing complex structural rearrangements during state changes. These events occur on a wide range of time and length scales, and no single method is sufficient to observe all of them. This thesis focuses on applying cryogenic electron microscopy to study the activation cycles of two target proteins.

The first part of the thesis work focuses on slow, large scale restructuring events in bacterial phytochromes. These red light sensing proteins can reversibly interconvert between two photochromic states via photoactivation, but they can also thermally relax to their resting state. Papers I-III present the structures of the two metastable states of the bacterial phytochrome PaBphP, establishing the structural basis of photoswitching; and investigate the kinetics of the thermal relaxation process both spectroscopically and structurally, uncovering the molecular mechanism of an allosteric regulatory effect.

The second part employs time-resolved cryogenic electron microscopy to study rapid, small scale events. Here, we present the development and application of an instrument capable of capturing structural intermediates forming on microsecond time scales. As proof-of-concept, the bacterial Ca2+ transporter LMCA1 is recorded 15 and 60 microseconds after initiating ATP binding, revealing the sequence of events leading to the ejection of the transported ion.