Critical role of tryptophanyl residues in the conformational stability of goose delta-crystallin.
Delta-crystallin is the major structural protein in avian and reptilian eye lenses but its sequence is highly homologous with the urea cycle enzyme, argininosuccinate lyase (ASL). In previous studies the multi-step unfolding process of this protein in the presence of GdmCl was sensitively probed using tryptophan fluorescence. In this study the contribution of single tryptophan residues to the stability of the local environment was monitored by mutation of two highly conservative tryptophan residues in goose delta-crystallin, Trp 74 and Trp 169. These residues behaved differently in terms of fluorescence intensity and maxima emission wavelength, consistent with their structural location in buried or solvent accessible regions. No gross changes in the secondary structure after mutation were observed, as judged by far-UV CD. The side chains of tryptophan residues in the structure of wild-type goose delta-crystallin possess both hydrophobic and hydrogen bonding interactions. Replacement of the side chain with phenylalanine or alanine led to expose of a hydrophobic area and a reduction in thermal stability; W169A particularly has a T(m) value that was 10 degrees C lower than the wild type enzyme. In the presence of GdmCl, a sharp red shift in fluorescence wavelength due to subunit dissociation can be sensitively detected using a single tryptophan, with the region surrounding W74 undergoing the first transition with a [GdmCl](1/2) of 0.45 M. Further measurement of unfolding curves by CD revealed that the W169A mutant was most unstable with a [GdmCl](1/2) of 0.22 M. From sedimentation velocity analysis, the unstable conformation of the W169A mutant affected the assembly of the quaternary structure. Our studies demonstrate the critical role for the tryptophan residues in stabilizing protein conformations and subunit assembly in delta-crystallin.
Lee
Hwei-Jen
HJ
Department of Biochemistry and Institute of Molecular Biology, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu, Taipei 114, Taiwan. hjlee@mail.ndmctsgh.edu.tw
Lai
Young-Hsang
YH
Huang
Yu-Ting
YT
Huang
Chih-Wei
CW
Chen
Yu-Hou
YH
Chang
Gu-Gang
GG