Folding of an all-beta protein: independent domain folding in gamma II-crystallin from calf eye lens.

Abstract

ABSTRACTyII-crystailin from calf eye lens consists of two homologous domains, each composed of two similar "Greek key" motifs. As a consequence of the bilobal structure, a biphasic transition is seen upon unfolding by urea at low pH (monitored by circular dichroism, fluorescence emission, and ultracentrifugal analysis). In 3.3 ± 0.5 M urea, a stable intermediate is formed at equilibrium, whereas 5.5 M urea causes maximum denaturation. Unfolding/folding kinetics display a complex pattern characterized by two kinetic phases. Both reactions exhibit strong dependence on the urea concentration; in the range ofthe respective transition, their rates are extremely slow (k = 1 x 10-4 s-'). In conclusion, folding of yHl-crystallin proceeds through the independent sequential structuring of the domains.Eye lens proteins are not subject to the general protein turnover; they remain in their native conformation during the whole life-span of the organism. The molecular mechanism underlying the extreme long-term stability is presently unknown. y-crystallins, the main components ofthe core region ofthe eye lens, have been characterized in detail: the amino acid sequences of most proteins from calf eye lens are known, and for yII-crystallin, the crystal structure has been solved at high resolution (1). The protein is a monomer of 20 kDa that does not contain disulfide bonds; it consists of two homologous domains, each composed of two "Greek key" motifs forming a sandwich of two four-stranded antiparallel a-pleated sheets (2, 3). Because 'yII-crystallin is almost entirely composed of ,l-sheets, this protein should be a good model for the analysis of P-structure formation.'yII-crystallin is a highly stable protein that preserves its native state at pH 1-10. In 0.1 M phosphate buffer, pH 7, the protein is stable to 75°C; at higher temperature, irreversible aggregation occurs. In 7 M urea, denaturation is only seen at elevated temperature or low pH; under these conditions, a two-step equilibrium transition suggests independent domain folding (4).We report here equilibrium and kinetic studies on the domain folding of yII-crystallin. Sedimentation analysis, intrinsic fluorescence, circular dichroism, and thermal analysis prove that the overall reaction at pH 2 can be quantitatively described by a three-state model: MATERIALS AND METHODSPreparation of yII-Crystallin and Its NH2-Terminal Domain. yII-crystallin from calf eye lenses was prepared according to the method of Bjork (5), modified by van Dam (6) and Nesslaier (7). Purity of the protein was ascertained by SDS/PAGE, isoelectric focusing, and spectroscopic techniques: Phast-system (Pharmacia), fluorescence spectrophotometer MPF 44A (Hitachi/Perkin-Elmer), spectropolarimeter J-500/DP-500N (Jasco, Easton, MD). Fast protein liquid chromatographic separations (Pharmacia) made use of the gradient programmer GP-250 with UV-1 monitor.To isolate the NH2-terminal domain fragment, partially unfolded yII-crystallin (obtained by 24-hr incubation of the protein in 3 M urea/0.1 M NaCl/HCl, pH 2....

Kinetics of translation of γB crystallin and its circularly permutated variant in an in vitro cell‐free system: possible relations to codon distribution and protein folding

“…The mutual stabilization of the domains is reflected by their enhanced stabilities in the full-length yBcrystallin compared to its separate domains; the corresponding AAGs~ab values for the N-and C-terminal domains are 9 and 15 kJ/mol, respectively. Similar differences are observed comparing the folding/unfolding kinetics [5]: The N-terminal domain folds significantly faster than the C-terminal one [2,4]. Taken together, previous findings suggest that the N-terminal domain serves as a core structure for the folding and stabilization of the C-terminal half of the molecule, thus generating the anomalously high stability of yB-crystallin.…”

“…However, in spite of the extreme symmetry of the molecule, its domains show significant differences in their stability against urea denaturation. Unfolding/folding experiments reveal sequential and independent domain folding, with the C-terminal domain showing relatively low stability and enhanced unfolding compared with the N-terminal one which requires extreme denaturant conditions to unfold [2,3] Abbreviations: yB, ~'C, 7CP and yN, natural ~,B crystallin, C-terminal domain of yB, circularly permutated yB, and N-terminal domain of yB, respectively; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; TCA, trichloroacetic acid.…”

Molecular basis of a progressive juvenile-onset hereditary cataract

“…Data were taken at pH 2 for comparison with previously published work on bovine ␥B crystallin (28,29). The increased sensitivity of our instrument enabled us to use 10-fold lower protein concentrations (0.1-0.2 mg͞ml) than those used by Kono et al (28) and Rudolf et al (29).…”

Equilibrium folding intermediates of a greek key β-barrel protein

“…The folding mechanisms of the eye lens bg-crystallins (Jaenicke, 1996), and gII-crystallin in particular (Rudolph et al, 1990), have been studied in some detail. gII-Crystallin is a two-domain protein in which each domain contains two Greek keys.…”