title: Configurational entropy modulates the mechanical stability of protein GB1.
abstract: Configurational entropy plays important roles in defining the thermodynamic stability as well as the folding/unfolding kinetics of proteins. Here we combine single-molecule atomic force microscopy and protein engineering techniques to directly examine the role of configurational entropy in the mechanical unfolding kinetics and mechanical stability of proteins. We used a small protein, GB1, as a model system and constructed four mutants that elongate loop 2 of GB1 by 2, 5, 24 and 46 flexible residues, respectively. These loop elongation mutants fold properly as determined by far-UV circular dichroism spectroscopy, suggesting that loop 2 is well tolerant of loop insertions without affecting GB1's native structure. Our single-molecule atomic force microscopy results reveal that loop elongation decreases the mechanical stability of GB1 and accelerates the mechanical unfolding kinetics. These results 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. This study not only demonstrates a general approach to investigating the structural deformation of the loop regions in mechanical unfolding transition state, but also provides the foundation to use configurational entropy as an effective means to modulate the mechanical stability of proteins, which is of critical importance towards engineering artificial elastomeric proteins with tailored nanomechanical properties.
authors.affiliation: Department of Chemistry, The University of British Columbia, Vancouver, BC, Canada. Hongbin@chem.ubc.ca
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
authors.forename: Hui-Chuan
authors.initials: HC
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Cao
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.forename: Meijia
authors.initials: M
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001696:Biomechanical Phenomena; D002942:Circular Dichroism; D019277:Entropy; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013291:Streptococcus / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus