title: Dissecting cooperative communications in a protein with a high-throughput single-molecule scalpel.
abstract: Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double-stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all-or-none and partially cooperative events, which represent strong and weak high-order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all-or-none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all-or-none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.
authors.affiliation: Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242 (USA); Current address: Department of Bionanoscience, Kavili Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft (The Netherlands).
authors.forename: Zhongbo
authors.initials: Z
authors.lastname: Yu
authors.forename: Yunxi
authors.initials: Y
authors.lastname: Cui
authors.forename: Sangeetha
authors.initials: S
authors.lastname: Selvam
authors.forename: Chiran
authors.initials: C
authors.lastname: Ghimire
authors.forename: Hanbin
authors.initials: H
authors.lastname: Mao
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D052898:Optical Tweezers; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D002147:Calmodulin; D004247:DNA
keywords: cooperative unfolding; hierarchical cluster analysis; high throughput; protein folding; single-molecule studies