Analysis of the three-dimensional structures of enzymes using X-ray crystallography is vital to our knowledge of how chemical reactions are carried out in cells. However, individual structures can provide only snapshots of complex and dynamic enzyme mechanisms. A team led by Dr Mike Hough from the University of Essex aims to generate three-dimensional ‘structural movies’ to illustrate the detailed dynamic functions of enzymes.
Since the 1950s, three-dimensional X-ray crystal structures of enzymes and other proteins have underpinned our understanding of biology. The speed and resolution with which these structures can be produced has greatly increased with successive generations of accelerator-based synchrotron X-ray sources. Individual crystal structures, however, can provide only discrete static steps of a dynamic enzyme mechanism, limiting their ability to describe dynamic and multi-step enzyme mechanisms. Essentially these structures may be considered as stills from a movie reel.
In this research project, utilising recent state of the art technology breakthroughs, we will overcome this limitation by creating multi-frame ‘structural movies’ illustrating the catalytic functions of enzymes at atomic resolution. This has been a dream of many for decades, currently only tractable via theoretical approaches. In brief, we will use X-rays to generate electrons within protein crystals. These electrons will migrate to redox centres related to the catalytic mechanism and be used to initiate and drive the enzyme catalytic cycles at will. Tools will be developed using emerging technologies (including ultrafast X-ray detectors allowing structures to be measured in rapid succession), to understand these processes and to generate structural movies for biologically important enzymes. Enzyme states generated in this way are representative of those occurring within living cells, where the electrons that drive reactions would be provided by biological electron transfer. Crystal spectroscopies will be used to unambiguously assign the different structural ‘frames’ to the correct step of the enzyme mechanism. These methods, initially used to test whether X-rays perturb the protein they are measuring, have yielded an important additional dividend which we are now able to exploit – the ability to identify states generated by X-ray-induced catalysis.
The interdisciplinary nature of the project and strong potential for training was a clear fit to the aims of the Leverhulme Trust; and will enable us to work closely with scientists at synchrotron laboratories to rigorously establish a novel methodology that is broadly applicable to the study of how enzymes carry out their many life-sustaining functions.
Dr Mike Hough
University of Essex
Dr Richard Strange
University of Liverpool