Study of protein structural disorder by electron paramagnetic resonance
Wilson, John C.
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Proteins do not adopt a unique, static structure; rather, they exist in a dynamic ensemble of conformations. In order to completely describe a protein structure, it is therefore necessary to quantitatively describe the amount of motion and disorder present. Although these features complicate the description of the protein structure, they often play important roles in protein function, and their description is therefore valuable. In this thesis, disordered and dynamic structural elements of two proteins are studied in solution using electron paramagnetic resonance (EPR), a site-specific magnetic resonance technique. Using this approach, we are able to quantify the amount of disorder and motion in specific structural elements which are themselves crucial for protein function. Quantitative analysis of disorder and motion allows us to gain insight into the role of specific structural elements within these proteins.;The active form of the cyclooxygenase domain in prostaglandin H 2 synthase-1 (PGHS-1) contains a tyrosyl radical which abstracts a hydrogen from the substrate to initiate turnover. PGHS-1 undergoes self-inactivation via an unknown mechanism which is thought to involve additional tyrosyl radicals. In this thesis, we use high frequency EPR and electron-nuclear double resonance to directly detect a hydrogen bond to the catalytically competent radical, suggesting that PGHS-1 self-inactivation is the result of radical transfer and not only a conformational change in the initial radical. Furthermore, we are able to characterize the geometry of this hydrogen bond, both identifying a hydrogen bond partner and demonstrating that there is disorder in the hydrogen bond, manifest as a distribution in bond lengths.;The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii is subject to allosteric regulation of substrate specificity. The dNTP products serve as both positive and negative regulators of ribonucleotide reduction, functioning to maintain the balance of the cellular dNTP pool. Several loops surrounding the dNTP-binding site have been proposed to mediate allosteric regulation, but a specific mechanism has not been identified. We have spin-labeled three of these loops and investigated their motion and position as a function of effector binding. Continuous-wave EPR demonstrates that these loops display highly restricted motion, even in the effector-free state. There are few changes in the motion of these loops upon the addition of dNTPs, and these are limited to the immediate vicinity of the dNTP-binding site. These results indicate that the allosteric regulation of substrate specificity in RTPR is not mediated solely by a change in the structural dynamics of these loops. Such a finding suggests that changes in the position of these loops are more likely to be responsible for allostery. We have also used double electron-electron resonance to measure spin-spin distances and determine the position of one of these loops relative to the main body of the protein. Our results indicate that there are small shifts in the position of this loop, but no long-range motions nor a major change in the structural disorder as measured by a distribution in interspin distances. It is therefore likely that these subtle structural changes are responsible for mediating allosteric regulation of substrate specificity in response to dNTP binding.;Both of the studies presented in this thesis represent an advancement in the application of existing EPR techniques for studying protein structures in solution. We show here that these applications have the ability to not only demonstrate the existence of motion and disorder in protein structures, but also to yield significant insight into the function of such motion and disorder.