␣-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of ␣-crystallin for aggregationprone proteins is probably based on hydrophobic interactions. ␣-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that ␣-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five ␣A-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of ␣A-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.The composition of the vertebrate eye lens is dominated by a group of structural proteins known as crystallins. The largest of these, ␣-crystallin, is a dynamic multimeric complex composed of two types of homologous subunits, ␣A-and ␣B-crystallin (1). A few years ago, it was discovered that these subunits are also constitutively expressed in various non-lenticular tissues, suggesting that their function is more than merely structural (2-5). In addition, increased expression of ␣B-crystallin has been observed in a variety of neurodegenerative disorders such as multiple sclerosis (6), Alexander's disease (7,8), and Alzheimer's disease (9, 10). Although the physiological significance of this extralenticular expression is unknown, it certainly relates to the fact that ␣-crystallins belong to the family of small heat shock proteins (hsp) 1 (11, 12). Among the shared features of ␣-crystallins and other small hsp are the conferring of thermotolerance (13,14), interaction with actin (15, 16), phosphorylation (17, 18), and intracellular relocalization upon stress (19,20). Furthermore, in vitro experiments have shown that ␣A-and ␣B-crystallin as well as hsp25 can act as molecular chaperones by suppressing aggregation of denaturing proteins (21-23). Unfortunately, the mechanism of this functional activity is unclear because the three-dimensional structure of ␣-crystallin is unknown.Native ␣-crystallins occur as polydisperse particles with an average molecular mass of about 800 kDa. Understanding the multimeric arra...

Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, AB-crystallin, but was less heat stable, denaturing upon heating to 50°C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone-like activity than AB-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1 H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.Keywords : small heat-shock protein; dimer ; oligomer; chaperone-like activity ; protein structure.In mammals, six members of the ubiquitous superfamily of small heat-shock proteins (sHsp) are known to occur [1]. These are AA-and AB-crystallin, Hsp25 (depending on species, also indicated as Hsp27 or Hsp28), the more recently discovered p20 [2], (now dubbed Hsp20 [3]), and the latest additions, HSPB2[4] and HspB3 [5]. A-Crystallin is a major eye lens protein, composed of two types of subunits, AA-crystallin and AB-crystallin (for reviews see [6,7]). The latter also occurs at high levels in other tissues, notably in heart and striated muscle [8]. AB-crystallin is stress-inducible [9] and its level is increased in various neurodegenerative disorders and tumors [10Ϫ13]. Hsp25 occurs at low levels in various tissues and is also inducible upon stress (for review see [14]). Both A-crystallins and Hsp25 display ATP-independent chaperone-like properties and confer thermotolerance upon expression in cultured cells. Mammalian A-crystallins and Hsp25 have been studied extensively. In contrast, Hsp20 has been the subject of only four reports [2,3,15,16], while HSPB2 and HspB3 are, as yet, the least known [4,5]. Hsp20 was originally isolated in mixed complexes with AB-crystallin and Hsp25 from rat and human skeletal muscle, and its amino acid sequence is most similar to AB-crystallin [1, 2]. Hsp20 was detected in all rat tissues examined, reaching the highest levels (up to 1.3 % of total protein) in striated muscle, heart and diaphragm, similar to AB-crystallin and Hsp25. Like Hsp25, it is also conspicuously present in smooth muscle of the bladder and rectum. Hsp20 exists in muscle extracts in a multimeric and a disso...

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.

The amine‐donor substrate specificity of tissue‐type transglutaminase has been studied in a series of recombinant αA‐crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C‐terminal extended arm of αA‐crystallin. A biotinylated amine‐acceptor hexapeptide was used as a probe for labelling the amine‐donor sites. Wild‐type bovine αA‐crystallin does not function as an amine‐donor substrate for tissue‐type transglutaminase. Yet, upon introduction of a lysine residue at the C‐terminal or penultimate position, all mutant αA‐crystallins act as amine‐donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine‐donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.

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