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研究セミナー Probing the hidden dynamics and energetics of diverse biomolecular systems by AFM

講演

Thomas T. Perkins[JILA, NIST & University of Colorado, Boulder, CO, USA.]

日時 2022年9月6日(火)14:00〜
場所 生命システム棟2F セミナー室 および Zoom配信(ハイブリッド)
世話人

上田昌宏(生命機能研究科)
Tel: 06-6879-4611
E-mail: masahiroueda[at]fbs.osaka-u.ac.jp

Atomic force microscopy (AFM)-based single-molecule force spectroscopy enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex unfolding pathway of individual membrane proteins. Such studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing the force stability, force precision, and time resolution of bioAFM. For an advanced, small-format commercial AFM, we uncovered how these three metrics were limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications using a focused ion beam that yielded enhanced data quality. High-precision single-molecule studies of membrane proteins—the target for over 50% of current and future drugs—have lagged analogous studies of globular proteins due to instrumental limitations. We revisited the unfolding of the canonical membrane protein bacteriorhodopsin with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The resulting data revealed the unfolding pathway in unprecedented detail. Toward the broader goal of measuring quantitative energetics, we leveraged these rapid and reversible dynamics to reconstruct the 1D free-energy landscape of bacteriorhodopsin’s initial unfolding to determine the free-energy change (ΔΔG0) for select point mutants. Hence, we established a platform for precisely quantifying membrane-protein energetics under native-like conditions. I will conclude by discussing extensions of this capability to other diverse biomolecules, including nucleic-acid structures and globular proteins involved in pathogenesis.


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