Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

Using Helix-coil Models to Study Protein Unfolded States

An abstract of a thesis devoted to using helix-coil models to study unfolded states.\\

Research on polypeptide unfolded states has received much more attention in the last decade or so than it has in the past. Unfolded states are thought to be implicated in various

misfolding diseases and likely play crucial roles in protein folding equilibria and folding rates. Structural characterization of unfolded states has proven to be

much more difficult than the now well established practice of determining the structures of folded proteins. This is largely because many core assumptions underlying

folded structure determination methods are invalid for unfolded states. This has led to a dearth of knowledge concerning the nature of unfolded state conformational

distributions. While many aspects of unfolded state structure are not well known, there does exist a significant body of work stretching back half a century that

has been focused on structural characterization of marginally stable polypeptide systems. This body of work represents an extensive collection of experimental

data and biophysical models associated with describing helix-coil equilibria in polypeptide systems. Much of the work on unfolded states in the last decade has not been devoted

specifically to the improvement of our understanding of helix-coil equilibria, which arguably is the most well characterized of the various conformational equilibria

that likely contribute to unfolded state conformational distributions. This thesis seeks to provide a deeper investigation of helix-coil equilibria using modern

statistical data analysis and biophysical modeling techniques. The studies contained within seek to provide deeper insights and new perspectives on what we presumably

know very well about protein unfolded states. \\

Chapter 1 gives an overview of recent and historical work on studying protein unfolded states. The study of helix-coil equilibria is placed in the context

of the general field of unfolded state research and the basics of helix-coil models are introduced.\\

Chapter 2 introduces the newest incarnation of a sophisticated helix-coil model. State of the art modern statistical techniques are employed to estimate the energies

of various physical interactions that serve to influence helix-coil equilibria. A new Bayesian model selection approach is utilized to test many long-standing

hypotheses concerning the physical nature of the helix-coil transition. Some assumptions made in previous models are shown to be invalid and the new model

exhibits greatly improved predictive performance relative to its predecessor. \\

Chapter 3 introduces a new statistical model that can be used to interpret amide exchange measurements. As amide exchange can serve as a probe for residue-specific

properties of helix-coil ensembles, the new model provides a novel and robust method to use these types of measurements to characterize helix-coil ensembles experimentally

and test the position-specific predictions of helix-coil models. The statistical model is shown to perform exceedingly better than the most commonly used

method for interpreting amide exchange data. The estimates of the model obtained from amide exchange measurements on an example helical peptide

also show a remarkable consistency with the predictions of the helix-coil model. \\

Chapter 4 involves a study of helix-coil ensembles through the enumeration of helix-coil configurations. Aside from providing new insights into helix-coil ensembles,

this chapter also introduces a new method by which helix-coil models can be extended to calculate new types of observables. Future work on this approach could potentially

allow helix-coil models to move into use domains that were previously inaccessible and reserved for other types of unfolded state models that were introduced in chapter 1.

Essays on Energy Economics and Industrial Organization

The dissertation consists of three chapters related to the low-price guarantee marketing strategy and energy efficiency analysis. The low-price guarantee is a marketing strategy in which firms promise to charge consumers the lowest price among their competitors. Chapter 1 addresses the research question "Does a Low-Price Guarantee Induce Lower Prices'' by looking into the retail gasoline industry in Quebec where there was a major branded firm which started a low-price guarantee back in 1996. Chapter 2 does a consumer welfare analysis of low-price guarantees to drive police indications and offers a new explanation of the firms' incentives to adopt a low-price guarantee. Chapter 3 develops the energy performance indicators (EPIs) to measure energy efficiency of the manufacturing plants in pulp, paper and paperboard industry.

Chapter 1 revisits the traditional view that a low-price guarantee results in higher prices by facilitating collusion. Using accurate market definitions and station-level data from the retail gasoline industry in Quebec, I conducted a descriptive analysis based on stations and price zones to compare the price and sales movement before and after the guarantee was adopted. I find that, contrary to the traditional view, the stores that offered the guarantee significantly decreased their prices and increased their sales. I also build a difference-in-difference model to quantify the decrease in posted price of the stores that offered the guarantee to be 0.7 cents per liter. While this change is significant, I do not find the response in comeptitors' prices to be significant. The sales of the stores that offered the guarantee increased significantly while the competitors' sales decreased significantly. However, the significance vanishes if I use the station clustered standard errors. Comparing my observations and the predictions of different theories of modeling low-price guarantees, I conclude the empirical evidence here supports that the low-price guarantee is a simple commitment device and induces lower prices.

Chapter 2 conducts a consumer welfare analysis of low-price guarantees to address the antitrust concerns and potential regulations from the government; explains the firms' potential incentives to adopt a low-price guarantee. Using station-level data from the retail gasoline industry in Quebec, I estimated consumers' demand of gasoline by a structural model with spatial competition incorporating the low-price guarantee as a commitment device, which allows firms to pre-commit to charge the lowest price among their competitors. The counterfactual analysis under the Bertrand competition setting shows that the stores that offered the guarantee attracted a lot more consumers and decreased their posted price by 0.6 cents per liter. Although the matching stores suffered a decrease in profits from gasoline sales, they are incentivized to adopt the low-price guarantee to attract more consumers to visit the store likely increasing profits at attached convenience stores. Firms have strong incentives to adopt a low-price guarantee on the product that their consumers are most price-sensitive about, while earning a profit from the products that are not covered in the guarantee. I estimate that consumers earn about 0.3% more surplus when the low-price guarantee is in place, which suggests that the authorities should not be concerned and regulate low-price guarantees. In Appendix B, I also propose an empirical model to look into how low-price guarantees would change consumer search behavior and whether consumer search plays an important role in estimating consumer surplus accurately.

Chapter 3, joint with Gale Boyd, describes work with the pulp, paper, and paperboard (PP&PB) industry to provide a plant-level indicator of energy efficiency for facilities that produce various types of paper products in the United States. Organizations that implement strategic energy management programs undertake a set of activities that, if carried out properly, have the potential to deliver sustained energy savings. Energy performance benchmarking is a key activity of strategic energy management and one way to enable companies to set energy efficiency targets for manufacturing facilities. The opportunity to assess plant energy performance through a comparison with similar plants in its industry is a highly desirable and strategic method of benchmarking for industrial energy managers. However, access to energy performance data for conducting industry benchmarking is usually unavailable to most industrial energy managers. The U.S. Environmental Protection Agency (EPA), through its ENERGY STAR program, seeks to overcome this barrier through the development of manufacturing sector-based plant energy performance indicators (EPIs) that encourage U.S. industries to use energy more efficiently. In the development of the energy performance indicator tools, consideration is given to the role that performance-based indicators play in motivating change; the steps necessary for indicator development, from interacting with an industry in securing adequate data for the indicator; and actual application and use of an indicator when complete. How indicators are employed in EPA’s efforts to encourage industries to voluntarily improve their use of energy is discussed as well. The chapter describes the data and statistical methods used to construct the EPI for plants within selected segments of the pulp, paper, and paperboard industry: specifically pulp mills and integrated paper & paperboard mills. The individual equations are presented, as are the instructions for using those equations as implemented in an associated Microsoft Excel-based spreadsheet tool.