alphaB-crystallin: a hybrid solid-state/solution-state NMR investigation reveals structural aspects of the...

Atomic-level structural information on alphaB-Crystallin (alphaB), 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 approximately 580-kDa human alphaB assembled from 175-residue 20-kDa subunits. An approximately 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 alphaB. In vitro, the chaperone-like activities of full-length alphaB 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 alphaB 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 alphaB. 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) (1)H-(15)N 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 alphaB 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).