C O M P L E X S Y S T E M S A N D B I O M E D I C A L S C I E N C E S
S C I E N T I F I C H I G H L I G H T S
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Visualising cooperative domain movements in an enzymatic reaction
Proteins frequently rearrange their structures to carry out their designated biological functions. These structural fluctuations are not random, but have evolved to follow carefully coordinated pathways, much like the moving parts in a combustion engine. A deeper understanding of structural biology requires the visualisation of dynamic events such as domain movements and unfolding in real time.
Real-time observation of sequential reaction dynamics requires a controlled trigger that can initiate the desired response. In addition, intermediate states in the reaction need to be populated to a large extent to enable experimental observation. If the protein of interest can be triggered to synchronously transit intermediate states in a reaction, X-ray pulses can then monitor the structural changes in the sample. Beamline ID09 is well suited for such pump- and-probe experiments thanks to the synchronisation of the exciting laser and probing X-ray pulses. In earlier work, the structures and kinetics of the intermediate states of a primary active membrane protein transporter involved
in the relaxation of skeletal muscles were identified . As adenosine 5 -triphosphate (ATP) provides the energy that fuels primary active transport, this first experiment relied on the laser-induced release of a photo-protected form of ATP to trigger the reaction. With this pump-and- probe methodology established at ID09, the path was open to investigate other ATP-dependent biological phenomena.
The adenylate kinase (AdK) protein catalyses phosphate- group transfer, and hence controls the composition of adenosine 5 -monophosphate (AMP), adenosine 5 -diphosphate (ADP), and ATP, thereby playing a central role in maintaining cellular energy homeostasis. Due to its relatively small size (23.6 kDa) and large-scale conformational rearrangements associated with the enzymatic reaction, AdK has become a model system to study the relationships between protein structure, conformational dynamics, and catalysis in enzymes. However, direct experimental evidence for the coordinated structural rearrangements upon ATP binding is still missing. Time-resolved X-ray solution scattering (TR-XSS) was used at ID09 to track the ATP-dependent response of AdK in real time. The advantage of the TR-XSS methodology over alternative biophysical structural characterisation
Fig. 57: TR-XSS data of ATP-dependent AdK
structural dynamics. a) Time-resolved
X-ray data, (b) time- independent basis
spectra, and (c) time evolution of population
densities of the identified transient states.