title: Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein.
abstract: Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding βγ-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding βγ-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca(2+), which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.
authors.affiliation: From the Program in Computational Biology and Bioinformatics, zns@duke.edu.
authors.forename: Zackary N
authors.initials: ZN
authors.lastname: Scholl
authors.affiliation: the Department of Mechanical Engineering and Materials Science, and.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: the Department of Chemistry, Duke University, Durham, North Carolina 27708 weitao.yang@duke.edu.
authors.forename: Weitao
authors.initials: W
authors.lastname: Yang
authors.affiliation: the Department of Mechanical Engineering and Materials Science, and pemar@duke.edu.
authors.forename: Piotr E
authors.initials: PE
authors.lastname: Marszalek
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force; D056004:Molecular Dynamics Simulation; D000072417:Protein Domains; D017293:Protein S / Q000737:chemistry / Q000378:metabolism; D038222:gamma-Crystallins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017293:Protein S; D038222:gamma-Crystallins; D002118:Calcium
keywords: atomic force microscopy (AFM); calcium; crystallin; molecular dynamics; protein folding