title: Limited proteolysis of gamma II-crystallin from calf eye lens. Physicochemical studies on the N-terminal domain and the intact two-domain protein.
abstract: Gamma II-crystallin from calf eye lens consists of two homologous domains, connected by a six-residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10-kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N-terminus and the C-terminal sequence showed that cleavage occurred at the Phe88-Arg89 peptide bond, giving rise to the complete N-terminal domain including the connecting hexapeptide. The C-terminal part of the polypeptide chain is cleaved to small fragments. Comparing the spectral properties of the isolated N-terminal domain and intact gamma II-crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 10-kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine . HCl is decreased, due to the lack of domain interactions stabilizing the intact protein. Unfolding/folding kinetics of the 10-kDa fragment coincide with the second phase of the bimodal transition of intact gamma II-crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule.
authors: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
authors: A K
authors: AK
authors: Sharma
authors: V
authors: V
authors: Minke-Gogl
authors: P
authors: P
authors: Gohl
authors: R
authors: R
authors: Siebendritt
authors: R
authors: R
authors: Jaenicke
authors: R
authors: R
authors: Rudolph
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014461:Ultracentrifugation; D014508:Urea
chemical_list: D003459:Crystallins; D014508:Urea