Characterizing Protein Conformations Using Copper-Based Pulsed Dipolar Esr Spectroscopy And Complimentary Biophysical Methods
Proteins play an important and diverse role in all living organisms. If proteins are unable to carry out their prescribed functions, the results can be problematic, or even fatal, for an organism. For example, in humans, Alzheimer's and Parkinson's are just 2 of the many diseases caused by proteins which either do not function, or function incorrectly. Proteins can also perform chemical transformations which are very difficult via synthetic methods, such as the oxidation of methane to methanol and the fixation of dinitrogen to ammonia. Understanding the mechanisms of these processes may lead to much more efficient catalysts, greatly reducing the large energy expenditures currently required. In biochemistry, the link between structure and function has been well established, and so in order to understand the mechanisms and functions of proteins, we must understand their structures. In many cases, the structures of flexible proteins can be difficult to elucidate, especially if multiple conformations exist simultaneously. Here, we use copper-based pulsed dipolar ESR spectroscopy (PDS) and other, complimentary biophysical and biochemical methods to characterize protein conformations in flexible proteins. These include mutants of Superoxide Dismutase 1 (SOD1) which cause familial ALS, and the Drosophila melanogaster circadian clock protein Period. By using these techniques, we show that fALS mutants of SOD1 tend to aggregate in solution as opposed to the wild-type (WT) protein which does not. Furthermore, we propose a structural mechanism by which this aggregation occurs. In the Period protein, we have discerned small differences in the conformation of mutants that mimic phosphorylation vs. the WT. These subtle changes may cause differences in circadian behavior observed in fruit flies.
Copper; ESR; Protein
Chen,Peng; Freed,Jack H
Chemistry and Chemical Biology
Ph.D. of Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis