This dissertation is a multidisciplinary approach to the understanding of the mechanical properties of proteins and their kinetics of unfolding, through the use of an atomic force microscope in force-clamp mode to perform single-molecule force spectroscopy. It begins with the development of a new experimental framework, which includes the design and construction of a new faster atomic force microscopy instrumentation, the definition of new bias-free experimental protocols, and the application of modern statistical tools for data analysis, all of these optimized for the use in force spectroscopy at the single molecule level. Through this new framework new insight was gained on the laws governing the mechanics of a protein in solution. The kinetics of unfolding of the protein ubiquitin were experimentally observed to feature a broad power-law distribution of unfolding rates, quality characteristic of glassy kinetics, which challenges the commonly accepted simplest single rate two-state model models for protein unfolding. Also, the transition between two highly stretched structural configurations of a protein was directly observed to be rate-limited, revealing the magnitude of the intrinsic friction limiting the speed of the first stage of protein folding. This document is presented with detailed technical and mathematical considerations with the intention to serve as handbook for the experimentalist interested in the study of polymers using force-clamp single molecule force spectroscopy.
|Adviser||Julio M. Fernandez|
|Subjects||Condensed matter physics; Biophysics|
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