Selectivity of Tryptophan Residues in Mediating Photolysis of Disulfide Bridges in Goat α-Lactalbumin

Abstract

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.

Photonic immobilization of bsa for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

“…Therefore, LAMI is used to immobilize BSA onto thiol derivatized slides as well as superparamagnetic particles aiming at the development of nanoparticles based drug delivery system and biosensors. Our earlier work as well as Vanhooren's work verified the crucial role of Trp on the photolytic cleavage of disulfide bridge using protein engineering (Petersen et al, 2006; Vanhooren et al, 2006). The estimated concentration of free thiols in BSA before and after 1 h 295 nm illumination was 0.35 and 0.98 µM, respectively.…”

Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein

“…1) or mAb-10F5 (data not shown). UV radiation likely disrupted the critical disulfide bonds required for generating high-affinity neutralizing epitopes as it is well-known that UV light can break disulfide bonds, especially through the photo-excitation of aromatic residues (Neves-Petersen et al, 2006;Vanhooren et al, 2006). In the past, UV-killed poxvirus vaccines were tested for the capacity to protect (Turner et al, 1970).…”

Novel chemical degradation pathways of proteins mediated by tryptophan oxidation: tryptophan side chain fragmentation

“…Mass spectrometry experiments have generated Trp +• in the gas phase, but interconversion to TrpN • (a distonic radical cation with protonated amino and carboxyl groups) was not observed, indicating a significant barrier for such process in the gas phase. In proteins, a close proximity of Trp to disulfide bonds can promote photo‐ionization, where disulfide bonds function as electron acceptors, undergoing reduction to disulfide radical anions (Scheme ; reactions 3 and 4), which can further dissociate into thiolate and thiyl radicals.…”