title: Folding of an all-beta protein: independent domain folding in gamma II-crystallin from calf eye lens.
abstract: gamma II-crystallin 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 of the respective transition, their rates are extremely slow (k approximately 1 x 10(-4)s-1). The kinetic mechanism of unfolding and refolding may be described by a three-state model: native in equilibrium intermediate in equilibrium denatured. The rate-determining steps are domain folding rather than domain pairing or proline isomerization. Kinetic analysis of the unfolding/folding of the intermediate populated in 3.0 M urea, pH 2.0, reveals that the kinetic and the equilibrium intermediates have similar structures. Limited proteolysis of gamma II-crystallin by pepsin in 3 M urea, pH 2, allows the NH2-terminal domain of the protein to be isolated. Unfolding/refolding of the fragment parallels the second transition in the above scheme, thus proving that the intermediate contains the COOH-terminal domain in its random state, whereas the NH2-terminal domain is still in its native conformation. In conclusion, folding of gamma II-crystallin proceeds through the independent sequential structuring of the domains.
authors.affiliation: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
authors.forename: R
authors.initials: R
authors.lastname: Rudolph
authors.forename: R
authors.initials: R
authors.lastname: Siebendritt
authors.forename: G
authors.initials: G
authors.lastname: Nesslaŭer
authors.forename: A K
authors.initials: AK
authors.lastname: Sharma
authors.forename: R
authors.initials: R
authors.lastname: Jaenicke
mesh_terms: D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007908:Lens, Crystalline; D008961:Models, Structural; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D003459:Crystallins; D014508:Urea