| This dissertation focuses on the effect of force on the mechanical properties of a wide range of biological systems using atomic force microscopy (AFM). These include protein unfolding and folding, variance analysis on the polysaccharide dextran, and probing the chemistry of enzymatic catalysis. Since single molecules can be stretched in AFM, it is possible to obtain large distributions, which can be used to develop, apply and test models from statistical mechanics.; A statistical analysis of protein unfolding under a constant force, which occurs in a two-state process, revealed that unfolding is not governed by a single rate-constant, as previously thought, but by a large range of constants spanning more than two decades. This, in turn, gave information about the underlying energy landscape, revealing that the protein in its folded state is trapped in many different conformations on a glassy, rugged energy landscape.; A similar analysis was applied to study the chemistry of enzymatic catalysis, where the enzymatic substrate was stretched under a constant force. By obtaining rates of catalysis at various forces and enzyme concentrations, we were able to deduce details of the underlying chemistry of the reaction. We detected two alternate pathways of catalysis and gained knowledge of their dynamic rearrangements during catalysis, which could not be probed with other current structural biology techniques.; My study on dextran explored the dynamics of its subunits, up to several hundred sugar rings, which, under a stretching force, flip from their equilibrium conformation to a more extended conformation. Variance analysis of this transition revealed that it is not a two-state process, as previously assumed, but has to occur through at least one intermediate.; Chapter 7 of this dissertation is devoted to protein folding. We found that protein collapse is mainly driven by hydrophobic interactions, and not by entropy, as commonly assumed. Based on these results, we proposed a folding energy landscape, which explains the so far conflicting results of protein folding with AFM and classical folding experiments. |
