Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC ) and human alphaB crystallin (ABC 68-162 ), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC 59-163 and ABC . A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.Keywords: X-ray diffraction; small heat shock protein; protein chaperone; desmin-related myopathy; cataract; eye lens transparency Abbreviations: AAC 59-163 , alphaA crystallin residues 59-163; AAC 59-163 -Zn, zinc-bound alphaA crystallin residues 59-163; ABC 68-162 , alphaB crystallin residues 68-162; ABC 68-157 , alphaB crystallin residues 68-157; ABC 67-157 , alphaB crystallin residues 67-157; ADH, alcohol dehydrogenase; AP, antiparallel beta sheet interface; AP x , antiparallel beta sheet registration state x; DS, domain-swapped interface; Hsp20 65-162 , rat heat shock protein 20 residues 65-162; MjHsp16.5, Methanococcus janaschii heat shock protein 16.5; MS, mass spectrometry; sHSP, small heat shock protein; WhHsp16.9, wheat heat shock protein 16.9.Additional Supporting Information may be found in the online version of this article.

ABSTRACT␣B-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in ␣B-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using ␣-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G ␣B-crystallin had reduced or completely lost chaperone-like function. The addition of R120G ␣B-crystallin to unfolding ␣-lactalbumin enhanced the kinetics and extent of its aggregation. R120G ␣B-crystallin became entangled with unfolding ␣-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G ␣B-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G ␣B-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G ␣B-crystallin has decreased ␤-sheet secondary structure and an altered aromatic residue environment compared with wild-type ␣B-crystallin. The apparent molecular mass of R120G ␣B-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type ␣B-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G ␣B-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of ␣B-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

␣-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each ␣-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant ␣-crystallin subunits. We labeled ␣A-crystallin with stilbene iodoacetamide (4-acetamido-4-((iodoacetyl)amino)stilbene-2,2-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled ␣A-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent ␣A-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min ؊1 at 37°C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several ␣A-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of ␣A-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.␣-Crystallin, the major lens protein of the mammalian eye, is a member of the small heat shock protein family (1, 2). Like other small heat shock proteins, ␣-crystallin is a high molecular mass complex consisting of a large number of subunits. The two polypeptides of ␣-crystallin found in the lens of the mammalian eye, ␣A and ␣B, are encoded by evolutionarily related genes and share more than 50% identity in amino acid sequence (3, 4). For many years, ␣-crystallin was thought to be lens-specific. However, recent advances in detection methods have revealed much wider non-lenticular tissue distributions in heart, thymus, skin, lung, retina, and brain (5-8).

A family of unusual proteins is deposited in flat, structural platelets in reflective tissues of the squid Euprymna scolopes. These proteins, which we have named reflectins, are encoded by at least six genes in three subfamilies and have no reported homologs outside of squids. Reflectins possess five repeating domains, which are highly conserved among members of the family. The proteins have a very unusual composition, with four relatively rare residues (tyrosine, methionine, arginine, and tryptophan) comprising approximately 57% of a reflectin, and several common residues (alanine, isoleucine, leucine, and lysine) occurring in none of the family members. These protein-based reflectors in squids provide a marked example of nanofabrication in animal systems.