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- Orädd, Fredrik, 1994-
(author)
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Determining the effects of regulatory parameters on the structural dynamics of P-type ATPase membrane transporters
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Proteins are macromolecular machines with roles in all cellular activities and structures. The functional properties of each protein is the result of its combination of 3D-structure and inherent dynamics, and a wealth of structural and dynamic mechanisms have evolved to regulate protein activity. P-type ATPases are membrane transport proteins that hydrolyze ATP to move cations across membranes. These proteins are involved in important biological functions such as Ca2+ signaling and Cu+ homeostasis, making proper regulation critical. Adenylate kinase (AdK) is a small, soluble protein that plays a role in energy homeostasis by interconverting ATP, AMP, and ADP, which are bound by two substrate binding domains. In this thesis, the effect of regulatory parameters on the structural dynamics of Cu+-ATPases and the sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) was investigated, together with the reaction dynamics of AdK.In Paper III, the human Cu+-ATPase ATP7B was simulated with (holo) and without (apo) Cu+ bound to the regulatory metal binding domains (MBDs, with MBD-1 closest to the core protein). In the holo state, the MBD chain was more dynamic and extended, and MBD-2 approached the membrane Cu+ entry site. In Paper IV, the stability of the interaction between MBD-2 and the Cu+-entry site was evaluated using MD simulations, showing that the interaction was stable in the cytosol-open E1 state, but not in the lumen-facing E2P state. An interaction site between MBD-3 and the cytoplasmic domains was also found, where MBD-3 might inhibit activity by interfering with functional motions. Finally, in Paper II, Cu+ entry into the membrane high-affinity Cu+-binding site was simulated, showing that a proposed initial binding site was transient and that the Cu+ ion could move deeper into the membrane domain. In Paper I, we used time-resolved X-ray solution scattering (TR-XSS) to show a simultaneous closing of the substrate binding domains in AdK, which included a partial unfolding and refolding event in the ATP-binding domain. Paper VI demonstrated that a novel time-resolved setup based on detector readout at the MAX IV beamline CoSAXS could trigger and detect AdK structural dynamics.In Paper V, TR-XSS experiments showed that the rate-limiting step in skeletal-muscle SERCA1a was an E1-to-E2P intermediate at both low and high Ca2+ concentrations. An inhibitory effect at high Ca2+ concentration was explained by a fraction of SERCA molecules stalling in the ATP-binding/phosphorylation step. In Paper VII, TR-XSS experiments showed that the housekeeping isoform SERCA2b, which is slower but has higher Ca2+ affinity than the other SERCA isoforms, shared the same rate-limiting step as the SERCA1a isoform, but with a longer rise-time. Deletion of the SERCA2b luminal extension (LE) shifted the rate-limiting step to ATP-binding/phosphorylation, possibly because of LE-stabilization of the ATP-bound structure. These papers demonstrated the capability of TR-XSS to detect changes in rate-limiting steps and to investigate how protein structural dynamics respond to mutations and inhibitory conditions.
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| 3. |
- Ådén, Jörgen, 1980-
(author)
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NMR studies of protein dynamics and structure
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.
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