Protein molecules are dynamic entities that explore complex energy landscapes when carrying out their biological functions. NMR spectroscopy is a powerful technique for defining the conformational states of proteins and for characterizing the dynamic processes in which multiple conformations interconvert with each other.  Our research aims at defining the functionally important conformational states of biomacromolecules not accessible by conventional structural biology tools. To accomplish the research goal, we integrate NMR spectroscopy with other biochemical and biophysical approaches. The ongoing projects are focused on the following three areas:

    • Effects of post-translational modifications on the structure and function of proteins
    • Functionally important intermediate states in protein-protein binding pathways
    • NMR method development

 Protein Motions in the Activation of Deubiquitinase A

      Deubiquitinase A (DUBA) was recently identified as a negative regulator of interferon-I (INF-I) production by cleaving the Lys63-linked poly-ubiquitin chains on TRAF3, which is an E3 ubiquitin ligase critical for INF-I production. Overproduction of INF-I has been linked to autoimmune diseases, such as systemic lupus erythematous. Crystal structures of apo DUBA and DUBA in complex with ubiquitin C-terminal aldehyde (Ub-al) revealed a disorder-to-order transition involving formation of helix 1 and helix 6 upon binding of Ub-al. Biochemical analysis indicated that phosphorylation of Ser177 is essential for activity, although structural changes were not observed upon phosphorylation.

    The proposed research will investigate how recognition of ubiquitin substrate is achieved by an interface that is largely disordered in the absence of substrate. The hypothesis is that the binding competent conformation pre-exists and upon binding the distribution of population of various conformers shifts toward the bound state from a wide structural ensemble. A second question of interest is how phosphorylation changes the distribution of conformers to activate the enzyme since the phosphorylation site Ser177 is located in the disordered region. Phosphorylation is a novel and largely uncharacterized post-translational modification mechanism for regulation of protease activity. Besides providing insights into DUBA, the proposed research can also lead to a mechanistic understanding of how phosphorylation regulates the activity of cysteine proteases.

    Besides DUBA, disordered regions are commonly observed in crystal structures of other deubiquitinase domains. This observation leads to the question of whether disordered structural components are essential for the catalytic cycle of deubiquitinases, especially the product release step. To answer this question, various mutants will be generated to stabilize the bound conformation and therefore reduce the conformational disorder in the unbound state. The dynamic properties and catalytic rates of these mutants will be determined and, if possible, quantitative relationship between the rate of conformational transitions and the rate of catalysis will be derived.

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