Protein Kinase A (PKA) plays a major role in the cell by acting as a key signaling hub in all eukaryotic cells. PKA is composed of a catalytic and regulatory subunit that combine to form an inactive complex. Binding of cAMP triggers a conformational change in the regulatory subunit that releases the active catalytic subunit. Mutations that affect the interaction between the regulatory and catalytic subunit result in the misregulation of PKA and consequently, in tumors that are associated with Cushing’s syndrome and Fibrolamellar Hepatocellular Carcinoma. The crucial involvement of PKA in cellular processes and disease emphasize the importance of understanding the basic mechanisms of activation and regulation of this protein. In this study, we use optical tweezers to selectively unfold a single regulatory subunit molecule alone or bound to the catalytic subunit. We find that the regulatory subunit unfolds at a greater force when bound to the catalytic subunit, indicating substantial surface interactions between the two subunits. Analysis of the unfolding force distribution shows two populations, suggesting two conformations in the PKA native state ensemble. A change in the unfolding axis, results in a highly cooperative dissociation of the PKA complex that is triggered by the perturbation of an energetic hub in the regulatory subunit. This study provides single molecule insight into the energetics between the regulatory and catalytic subunit that drive the formation of the PKA complex.

Identification of the molecular origin of disease with single molecule optical tweezers

254th National Meeting (2017)

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