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.
Highlights
AB-crystallin is an oligomeric vertebrate small heat-shock protein with molecular chaperone activity [1]
The model predicts that the basic monomeric structure is identical in all oligomers, which is confirmed by NMR experiments [25,31], and that the dimer interface is labile, as suggested by our results on the core domain and previous measurements [23,68,73]. Through fitting this model to the mass spectrometry (MS) data, the oligomeric distributions can be reproduced accurately, and the association free energies of the edge and dimer interfaces (DGd, and DGe, respectively) quantified [65]. To complement these thermodynamic parameters, the rate constants that govern the inter-conversion of the aBcrystallin oligomers can be determined using subunit exchange reactions in which the protein is incubated with a labelled counterpart [74]
We have presented a detailed investigation on the influence of the palindromic C-terminal residues on the thermodynamics and kinetics of aB-crystallin oligomers
Summary
AB-crystallin is an oligomeric vertebrate small heat-shock protein (sHSP) with molecular chaperone activity [1]. X-ray crystallography studies of N- and C-terminally truncated constructs of both aB-crystallin and its eye-lens-specific isoform aA [21 –24] as well as solid-state nuclear magnetic resonance spectroscopy (ssNMR) of the full-length protein [25] have revealed the structure of a dimeric ‘building block’ This protomer is composed of a b-sandwich ‘a-crystallin’ core that assembles through anti-parallel (AP) pairwise interactions between extended b6 þ 7 strands (figure 1). The crystal structure of a construct of aB-crystallin, truncated of the extension in addition to the N-terminal region, formed a runaway domain-swapped polymer, with the IXI lodged in the b4– b8 groove (figure 1b) [23] This binding of the IXI was demonstrated to pertain to the fulllength protein in ssNMR performed at low temperature [25]. The data were fitted to our oligomerization model [65], allowing the rate constants of dissociation k2e and k2e þ d, and rate of association kþ[aB1] to be extracted (see the electronic supplementary material, table S1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Philosophical Transactions of the Royal Society B: Biological Sciences
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
