In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state.
When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.
Subramaniam
V
V
University of Michigan, Ann Arbor 48109, USA.
Steel
D G
DG
Gafni
A
A
D000818:Animals; D002417:Cattle; D019791:Guanidine; D006146:Guanidines; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014801:Vitamin A / Q000378:metabolism
D006146:Guanidines; D007782:Lactoglobulins; D014801:Vitamin A; D014364:Tryptophan; D019791:Guanidine