Interaction of 1,1′-Bi(4-anilino)naphthalene-5,5′-Disulfonic Acid with α-Crystallin

Abstract

␣, ␤-, and ␥-crystallins constitute the major portion of the eye lens fiber cells (1). Among the crystallins, ␣-crystallin is the most abundant protein, existing as a polydisperse aggregate with the average molecular mass of 800 kDa (2). ␣-Crystallin is made up of two types of subunits, designated ␣A and ␣B with molecular masses 19,832 and 20,079 kDa, respectively (2). The sequences of the subunits of ␣-crystallin have high homology to small heat shock proteins (3, 4). ␣-Crystallin subunits, once thought to be lens-specific, are now widely known to be present in other tissues as well (5-8), and increased expression of ␣B-crystallin has been documented in some neurological disorders (6, 9, 10).Recently, the ability of native ␣-crystallin to suppress the aggregation of heat-denatured (11-26), 27), and chemically denatured (28) proteins and enzymes has been demonstrated. Complex formation between ␣-crystallin and denatured proteins and enzymes or ␤-and ␥-crystallins has been demonstrated (14, 18). On the basis of these in vitro data, it has been proposed that ␣-crystallin acts as a chaperone in vivo to maintain the lens clarity and that ␣-crystallin loses this ability during aging. Consistent with this hypothesis, a decreased chaperone-like activity has been observed for the ␣-crystallin present in high molecular mass aggregates from aged bovine and human lens (29,30).It has been proposed that surface hydrophobic sites in the native ␣-crystallin aggregate are involved in binding of target proteins to ␣-crystallin during chaperone-like activity display (17). A direct correlation between the extent of ␣-crystallin hydrophobicity and chaperone-like activity has been demonstrated (31-34). Liang and co-workers (35) in their recent study used recombinant ␣A-and ␣B-homopolymers and reported that the relative fluorescence enhancement of ANS 1 is greater with ␣B compared with ␣A and concluded that ␣B has higher hydrophobicity. However, so far the amino acid sequences that contribute to the hydrophobic site(s) have not been identified. In a recent report, Smulders and de Jong (36) described that the N-terminal domain of recombinant murine ␣B-crystallin binds hydrophobic probe bis-ANS. We have recently reported that amino acid residues 57-69 and 93-107 of ␣B-crystallins interact with heat-denaturing alcohol dehydrogenase (37). Liang and Li (38) reported that there are about 40 ANS binding sites/native ␣-crystallin. Stevens and Augusteyn (39) have disputed the study of Liang and Li and reported that there is one ANS binding site/24 subunits of ␣-crystallins. It is rather difficult to explain the stoichiometry of ANS binding to ␣-crystallin in view of the proposed complex but ordered structure for ␣-crystallin (2).In the present study we have determined the binding of bis-ANS to ␣-crystallin by equilibrium dialysis. The data presented here show the binding of bis-ANS to both A-and Bsubunits of ␣-crystallin and transfer of the energy from protein tryptophan to the bound fluorophore. Furthermore, we show that prior binding of bi...

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

“…5 and 6). ␣-Crystallin has exposed hydrophobic pockets in the absence of ATP that binds to bis-ANS (12,15,36,37). The substrate protein ␥-crystallin or carbonic anhydrase binds much less bis-ANS.…”

“…But when it binds to the hydrophobic sites of protein, its quantum yield dramatically increases (12). This dye has been widely used for probing the surface-exposed hydrophobicity of ␣-crystallin (12,15,36,37). We have done fluorescence titration of various ␣-crystallins by bis-ANS both in the presence and the absence of 3 mM ATP.…”

Crystallin proteins and amyloid fibrils

“…Hydrophobic sites in α-crystallin are thought to be responsible for its chaperone activity [45,[134][135][136][137] and hydrophobic peptides isolated from αA-crystallin (residues 70-88) [137] and αB-crystallin (residues 73-92 and 131-141) [24, 138] possess chaperone activity against amorphously aggregating target proteins. The chaperone activity of these peptides results from their ability to bind independently to target proteins [137].…”

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX

“…Exposure of hydrophobic domains was analyzed by measuring bis-ANS fluorescence with a SFM25 spectrofluorometer (Kontron). The excitation wavelength was set at 380 nm, and the emission was scanned between 400 and 600 nm (33). For the analysis of intrinsic tryptophan fluorescence, AtTDX (100 g/mL) incubated at various temperatures for 30 min was scanned after setting the excitation wavelength to 295 nm.…”