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Mechanical folding/unfolding of proteins probed by single molecule atomic force microscopy

University of British Columbia

The mechanical folding/unfolding of proteins is involved in many biological processes. However, the molecular mechanism underlining the mechanical folding/unfolding of proteins remains an open question. Most of the current knowledge about the protein folding is from ensemble measurements. In the study of the molecular mechanism underlying the mechanical folding/unfolding of proteins, single molecule atomic force microscopy (AFM) has its unique advantages. Although many endeavors have been made by using single molecule AFM to study the mechanical folding/unfolding of proteins and numerous interesting details have been revealed, the underlying mechanism of protein mechanical folding/unfolding remains largely unknown. The main objective of this thesis is to study the mechanical folding/unfolding of some model proteins using single molecule AFM. First, we studied the mechanical unfolding pathways of two domain-insertion proteins: a natural one, T4-lysozyme (T4L), and an artificially designed one, GL5/T4L (GL5: a mutant of protein GB1). Our study on T4L provided the first direct evidence of the kinetic partitioning assumption for protein folding at the single molecule level. Our study on GL5/T4L revealed its mechanical unfolding pathway with a reversed mechanical unfolding hierarchy. The designing of domain-insertion proteins also presented a new concept to program the mechanical unfolding pathway of multi-domain proteins. Second, we studied the mechanical folding/unfolding of TNfn3 domain by combining single molecule AFM with the steered molecular dynamics (SMD) simulation and protein engineering. The mechanical design of TNfn3 was found robust and the backbone H-bonds of TNfn3 were found critical for its mechanical stability. Our results showed the first direct evidence that the mechanical folding pathways of TNfn3 are governed by kinetic partitioning. Third, we studied the folding/unfolding kinetics and mechanics of an artificially designed mutually exclusive protein GL5/I27w34f (I27w34f: a tryptophan removed mutant of I27). The mutually exclusive protein GL5/I27w34f is designed to mimic the natural domain-insertion proteins which are typically difficult to study directly. Our study provided the first direct evidence that protein folding can generate sufficient mechanical strain to unravel a host protein and the folding of mutually exclusive proteins involving a tug-of-war. Mutually exclusive proteins provide a new system for manipulating protein folding.

Text

2011-04-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/33834

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/