Characterization of ATP-dependent protein dynamics under native-like conditions
Sammanfattning: Proteins are biological macromolecules capable of accelerating biochemical reactions. To accomplish this, proteins undergo changes in their molecular structure. Advances in structural biology have resulted in ever-increasing numbers of high-resolution protein structures. However, the majority of transient intermediate states will not amendable with traditional structural determination methods. Therefore, understanding how protein structural changes are correlated with the biological function necessitates development of methods that characterize the reaction in the native environment. P-type ATPase membrane transporters and the adenylate kinase (AK) are two ATP-dependent proteins that undergo extensive conformational change in their reaction cycles. While P-type ATPases maintain concentration gradients of ions across the cellular membranes, AK regulates cellular energy homeostasis by catalyzing interconversion of nucleotides. Resolving P-type ATPase and AK temporal and spatial structural dynamics is crucial to understand how these proteins are triggered by ATP for functionality. To pave way for time-resolved X-ray characterization of ATP-dependent conformational changes, it was necessary to identify optimal conditions for triggering protein reactions. Therefore, time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy of a recombinant Zn2+-transporting ATPase was used to optimize activation by photolysis of caged ATP. These conditions were then used to track structural dynamics of the Ca2+-transporting sarcoplasmic reticulum ATPase (SERCA) in skeletal muscle native membranes. Fast single-cycle dynamics were registered with the formation of an intermediate state at 1.5 ms followed by steady-state accumulation at 13 ms. The molecular dynamic (MD)-based structural refinement procedure showed that the 13-ms transient intermediate represented an ADP-sensitive, phosphorylated Ca2+-bound E1 state (Ca2E1P), with a domain arrangement that has so far eluded structural characterization.MD simulations of the identified SERCA transient intermediates further finetuned their positions in the reaction cycle. The 1.5-ms state was assigned to an ATP-bound state prior to phosphorylation, while the 13-ms state was stable in its Ca2E1P conformation. Because the simulations were performed in multicomponent lipid bilayers mimicking the native membrane, specific state-dependent lipid interactions were also identified. Finally, the wider applicability of the time-resolved X-ray method to study ATP-dependent protein dynamics was demonstrated by tracking AK structural dynamics. A transient intermediate at 5 ms was identified that showed closing of the ATP-binding domain prior to the NMP-binding domain, in the presence of both ATP and AMP substrates. This study provided conclusive experimental proof of the relative ordering of domain closure that had been predicted by several computational studies.In summary, the work presented in this thesis has contributed to developing the time-resolved X-ray method to study the structural dynamics of ATP-dependent proteins.
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