Comparison of the kinetics of S‐S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme

Abstract

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism~CD!, fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and0or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.

Photoinduced fibrils formation of chicken egg white lysozyme under native conditions

“…Without mutations, the formation of fibrillar aggregates of wild‐type CEWL under native conditions is generally impossible, because the native state of CEWL is quite stable with a conformational stability of 10.2 kCal/mol 48. Structural studies have highlighted the importance of these disulfide bonds to the stability of native structure of CEWL 49, 50. Here, we applied controlled UV doses to CEWL to selectively disrupt its terminal disulfide bonds and make its conformational states accessible from native state through thermal fluctuation under native condition.…”

The role of disulfide bond formation in the conformational folding kinetics of denatured/reduced lysozyme

“…Till now, the oxidative folding of lysozyme has been well studied, including its disulfide folding pathway [14][15][16] and folding kinetics [17][18][19][20][21]. However, these studies revealed little about what are the rate determinants of different phases.…”

Influence of the Internal Disulfide Bridge on the Folding Pathway of the CL Antibody Domain

“…[11][12][13][14] Accordingly, genetically engineered disulfide bonds have been used successfully as a tool to probe nucleation sites of the protein folding process. 5,[15][16][17] Additionally, a number of studies dealt with the formation of disulfide bridges within the folding process. [18][19][20][21][22] However, the influence of naturally occurring disulfide bridges on the mechanism of a protein folding reaction is still not completely understood even though theoretical considerations suggest it to be of pronounced importance.…”