Date of Award

Spring 1-1-2011

Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry & Biochemistry

First Advisor

Natalie G. Ahn

Second Advisor

Marcelo C. Sousa


Hydrogen exchange mass spectrometry (HX-MS) is an experimental technique that can be used to examine solvent accessibility and conformational mobility in biological macromolecules. HX-MS is well suited to probe protein solvent accessibility and conformational mobility, which can directly relate to cellular behavior and regulation. This thesis summarizes studies using HX-MS as the primary experimental technique to examine the regulated motions of MAP kinase and their role in protein activation. MAP kinases are a conserved family of proteins that are important for cellular regulation and proliferation that share a conserved secondary and tertiary structure and are activated by dual phosphorylation. The first study examines p38fÑ MAPK to see how changes in regulated motions upon activation compare between closely related family members. Structural analysis such as proteolysis, solvent accessibility, crystal structure details and dimerization are investigated and compared to earlier work examining ERK2 MAPK. The results reveal that even closely related proteins show vastly different changes in regulated motions upon activation. This work also touches on experimental strategies and methods for optimizing data recovery and resolution which have become possible due to the improvement of instrumentation and increased popularity of HX-MS. A second study built on past research and investigated changes in the hinge region of ERK2, previously identified as a region of interest. These studies examine the role the hinge plays in domain closure, substrate binding, catalysis and the changes that take place upon activation. Hinge mutants were created to increase flexibility to help determine the role the hinge plays in domain closure which is known to be important in the formation of a competent active site. The results suggest that enhanced hinge flexibility upon phosphorylation is needed for domain closure. In addition there is preliminary evidence that some residues in the hinge are important for ATP binding affinity. Lastly, the flexibility in the hinge leads to minimal increase in catalytic activity, indicating more than hinge flexibility is crucial for the increase in activity.