A protein fragment (M(r) ~9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% α-helix, 33% β-structure, and 28% remainder) according to the far-ultraviolet circular dichorism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristic of a globular organization. A single- step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, ΔG° (H2O), was found to be 10.80 ± 0.25 kJ/mol at 20 °C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 °C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the α- crystallin B chain. A search for proteolysis sites along the α-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N- terminal region. In conclusion, our findings suggest that the fragment can reasonably be identified as the free N-terminal domain of the α-crystallin B chain generated by in vivo hydrolysis. It folds autonomously both in vitro and in vivo independently of the native C-terminal extension. The structural stability has probably favored its accumulation in the degradation pathway of the α-crystallin B chain. A physiological role for the fragment has also been postulated.
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