title: Stability, homodimerization, and calcium-binding properties of a single, variant betagamma-crystallin domain of the protein absent in melanoma 1 (AIM1).
abstract: AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.
authors.affiliation: Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India.
authors.forename: Bheemreddy
authors.initials: B
authors.lastname: Rajini
authors.forename: Caroline
authors.initials: C
authors.lastname: Graham
authors.forename: Graeme
authors.initials: G
authors.lastname: Wistow
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Sharma
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003459:Crystallins; D018076:DNA, Complementary; D019281:Dimerization; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
chemical_list: C105445:CRYBG1 protein, human; D003459:Crystallins; D018076:DNA, Complementary; D008565:Membrane Proteins; D011506:Proteins; D002118:Calcium