Biomolecular Sciences Seminar Series: Dr. Thomas T. Perkins

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Biomolecular Sciences Seminar Series

Dr. Thomas T. Perkins, JILA, NIST & University of Colorado

Title: The hidden dynamics and energetics of bacteriorhodopsin and β-cardiac myosin probed by AFM

Abstract: Protein folding and dynamics occur as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states go undetected because of limited instrumental time resolution, force precision, or stability. We uncovered how each of these metrics in atomic force microscopy (AFM) was limited by the cantilever itself and overcame them using focused-ion-beam-modified cantilevers. We then revisited the unfolding of bacteriorhodopsin (bR), a canonical membrane protein embedded in its native lipid bilayer, achieving a 100-fold improvement in time resolution and a 10-fold improvement in force precision. Numerous newly detected intermediates—many separated by as few as 2–3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies lasting <10 &micro;s. We also exploited bR’s rapid, reversible initial unfolding to measure &Delta;&Delta;G for selected point mutants and, using sub-millisecond illumination, to further quantify the energetic consequence of a light-induced transition in the bR photocycle. I will conclude by discussing how these advances in bioAFM can be extended to human &beta;-cardiac myosin, the molecular motor that powers heart contraction. By combining single-molecule force spectroscopy with coarse-grained Brownian dynamics, we uncovered a previously unrecognized 2-nm mechanical transition in the myosin ankle—a junction that links lever-arm mechanics to ON/OFF-state regulation and thus to the number of heads available for contraction. This transition has implications for hypertrophic cardiomyopathy, the most common inherited heart disease.