Structural and kinetic characterization of the simplified SH3 domain FP1

The simplified SH3 domain sequence, FP1, obtained in phage display selection experiments has an amino acid composition that is 95% Ile, Lys, Glu, Ala, Gly. Here we use NMR to investigate the tertiary structure of FP1. We find that the overall topology of FP1 resembles that of the src SH3 domain, the hydrogen-deuterium exchange and chemical shift perturbation profiles are similar to those of naturally occurring SH3 domains, and the 15N relaxation rates are in the range of naturally occurring small proteins. Guided by the structure, we further simplify the FP1 sequence and compare the effects on folding kinetics of point mutations in FP1 and the wild-type src SH3 domain. The results suggest that the folding transition state of FP1 is similar to but somewhat less polarized than that of the wild-type src SH3 domain

Prediction and structural characterization of an independently folding substructure in the src sh3 domain

Previous studies of the conformations of peptides spanning the length of the a-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identi®ed a seven residue peptide in the src SH3 domain predicted to adopt a nativelike structure, a type II b-turn bridging unpaired b-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization con®rmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHa coupling constant patterns were indicative of a type II b-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-con®dence I-sites predictions identify protein segments that are likely to form native-like structures early in folding. # 1998 Academic Pres

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

Atomic-level structural information on αB-Crystallin (αB), 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 ∼ 580-kDa human αB assembled from 175-residue 20-kDa subunits. An ∼ 100-residue α-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and Cβ resonances have been obtained for residues 64–162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-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 α-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 αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) 1H–15N heteronuclear single quantum coherence spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer–monomer transition over the pH range 7.5–6.8. This steep pH-dependent switch may be important for αB 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)

Pharmacological chaperone for α-crystallin partially restores transparency in cataract models

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins

Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers.

The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry