title: C-terminal interactions mediate the quaternary dynamics of αB-crystallin.
abstract: αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins.
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
authors.forename: Gillian R
authors.initials: GR
authors.lastname: Hilton
authors.forename: Georg K A
authors.initials: GK
authors.lastname: Hochberg
authors.forename: Arthur
authors.initials: A
authors.lastname: Laganowsky
authors.forename: Scott I
authors.initials: SI
authors.lastname: McGinnigle
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Baldwin
authors.forename: Justin L P
authors.initials: JL
authors.lastname: Benesch
mesh_terms: D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013816:Thermodynamics; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism*
chemical_list: D038203:alpha-Crystallin B Chain