αB-Crystallin: A Hybrid Solid-State/Solution-State NMR Investigation Reveals Structural Aspects of the Heterogeneous Oligomer

Atomic-level structural information on alpha B-Crystallin (alpha B), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an similar to 580-kDa human alpha B assembled from 175-residue 20-kDa subunits. An similar to 100-residue alpha-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different alpha-crystallin domain constructs isolated from alpha B. In vitro, the chaperone-like activities of full-length alpha B and the isolated alpha-crystallin domain are identical. Chemical shifts of the backbone and C-beta resonances have been obtained for residues 64-162 (alpha-crystallin domain plus part of the C-terminus) in alpha B and the isolated alpha-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six beta-strands in the alpha-crystallin domain. A majority of residues in the alpha-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the alpha-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within alpha B. Evidence for a novel dimerization motif in the human alpha-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) H-1-N-15 heteronuclear single quantum coherence spectra as a function of pH. The isolated alpha-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for alpha B to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis). (C) 2008 Elsevier Ltd. All rights reserved.