“ESI-MSBioinformatics Studies on Crosslinking of αA-Crystallin and Lysozyme using a New Small Aryl Azido-N-HydroxySuccinimidyl Heterobifunctional Crosslinker based on a Metabolite of the Alternative Kynurenine Pathway”.

Abstract

The use of a new small aryl azido-N-Hydroxysuccinimidyl heterobifunctional crosslinker for crosslinking of αAcrystallin and lysozyme is described here. The crosslinker is based on the small molecule, 3-hydroxy anthranilic acid (3HAA) a part of the kynurenine pathway in Tryptophan metabolism. Enhanced amounts of 3HAA are found in disease states in the human body. The new crosslinker contains a photo labile azido group and an amine reactive, N-hydroxy succinimide (NHS) group. Small crosslinkers capture interacting protein interfaces better, while the larger ones are more useful for identifying interacting partners. Our earlier work has shown that aryl azides in this series lead to 'long lived' transients allowing for increased intermolecular reaction rates, otherwise difficult to achieve. Using this crosslinker, successful crosslinking of αA-Crystallin & lysozyme has been demonstrated in two steps i. e. incubation followed by photolysis (366 nm, 6W UV lamp). Previous studies on αA-Crystallin have mostly used only homobifunctional crosslinkers. As hypothesized by us, the use of a heterobifunctional crosslinker has indeed led to more efficient crosslinking. This has been confirmed using SDS-PAGE, ESI-MS/MS (following trypsinization of the homo and hetero 'dimer' bands) and use of StavroX 3.6.0.1, the bioinformatics software especially suited for analyzing intermolecular crosslinking. These investigations are expected to lead to a better understanding of the role of αA-Crystallin in chaperoning mechanism and in cataractogenesis.

A Multidimensional Approach to Explore the Use of a Small Heterobifunctional Crosslinker based on a Metabolite of the Kynurenine Pathway

“…Structural bioinformatics have been used extensively to explore the residue wise key information with and without small molecules, and StavroX 3.6.0.1 is particularly useful for identifying intermolecular crosslinking of proteins. Such intermolecular crosslinking has been carried out using a small heterobifunctional crosslinker [53,54] based on an essential amino acid Tryptophan a metabolite of the kynurenine pathway. This was done using the protocol Incubation, Photolysis, SDS-PAGE, excision of 'dimeric'band, trypsinization, ESI-MS, MS/ MS. Work described here shows that identical results are obtained by the use of two different bioinformatics software and a more refined 3D representation of the interfaces is obtained.…”