Single-molecule force-spectroscopy reveals the calcium dependency of the alternative conformations in the native state of a βγ-crystallin protein
Zackary Nathan Scholl, Qing Li, Weitao Yang and Piotr E. Marszalek*
- Author Affiliations
Duke University, United States
↵* Corresponding author; email: firstname.lastname@example.org
Author contributions: All authors helped design experiments. ZNS and QL performed the experiments. ZNS analyzed the data. All authors helped to write the manuscript.
To better understand the structure and stability of Protein S, researchers slowly pulled it apart. The red protein is Protein S and the blue proteins are used as "ties" for each end of the protein.
Source/Fonte: Duke University
Though multidomain proteins predominate the proteome of all organisms, and are expected to display complex folding behaviors and significantly greater structural dynamics as compared to single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands is poorly understood due to lack of experimental techniques. The multidomain calcium-binding β γ-crystallin proteins are particularly important, as their deterioration and misfolding/aggregation is associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding β γ-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 (SMFS) show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca2+, 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 high-charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.
atomic force microscopy (AFM) calcium crystallin molecular dynamics protein folding
Received March 28, 2016. Accepted July 4, 2016.
Copyright © 2016, The American Society for Biochemistry and Molecular Biology
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