Item – Theses Canada

​
OCLC number
1032890803
Link(s) to full text
LAC copy
Author
Wang, Hui-Chuan.
Title
Studies on the mechanical stability of a protein by single-molecule atomic force microscopy.
Degree
Master of Science - MSc -- University of British Columbia, 2009
Publisher
Vancouver : University of British Columbia, 2009.
Description
1 online resource
Notes
Includes bibliographical references.
Abstract
Elastomeric proteins are subject to mechanical tensions under biological settings and possess mechanical properties that underlie the elasticity of natural adhesive, cell adhesion and muscle proteins. Single molecule atomic force microscopy has made it possible to directly probe the mechanical properties of elastomeric proteins and provides insights into the molecular design of elastomeric proteins. Combining the single molecule atomic force microscopy and protein engineering techniques allows us study the mechanical stability of proteins and develop methods to tune the mechanical stability. Mechanical tensions are also found in some nonmechanical proteins. Based on the results from single-molecule atomic force microscopy, nonmechanical protein of GB1 shows high mechanical stability that is comparable or superior to those of known elastomeric proteins. Here, we use a small protein, GB1, the B1 IgG binding domain of protein G from Streptococcus, as a model system to directly investigate the mechanical properties of GB1 mutants and loop mutants by using single-molecule AFM. Point mutations in proteins may disrupt the intermolecular interactions and affect the chemical and mechanical stability of the protein. Φ-value analysis together with single-molecule atomic force microscopy is used to probe the mechanical stability of the protein and gives a complete picture on how proteins are structured in the transition state during folding/unfolding event. Results from chapter 2 indicate that GF30L and GT53A mutation decrease the mechanical stability as well as accelerate the unfolding kinetics of GB1. This is due to the disruption of the hydrogen bond networking between the terminal [beta]-strands or unraveled the hydrophobic interactions and side chain interactions, resulting in lower unfolding forces with Φ-values closer to one. Configurational entropy plays crucial roles in defining the thermodynamic stability as well as the folding/unfolding kinetics of proteins. Here, we directly probe the role of configurational entropy in the mechanical unfolding kinetics and mechanical stability of proteins by using single molecule atomic force microscopy and protein engineering methods. Chapter 3 demonstrates that the mechanical stability of GB1 decreases as the number of inserted amino acid residues into loop 2 of GB1 increases. This result can be explained by the loss of configurational entropy upon closing an unstructured flexible loop using classical polymer theory, highlighting the important role of loop regions in the mechanical unfolding of proteins. The findings from these experiments are of critical importance towards engineering artificial elastomeric proteins with tailored nanomechanical properties.
Other link(s)
hdl.handle.net
circle.ubc.ca