Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study

Abstract

Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern−Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn−Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.

Advances and prospects of analytic methods for bacterial transglycosylation and inhibitor discovery

“…99,101 Other possible mechanisms such as photoinduced electron transfer, 102 static and collisional interactions are also reported. 101,103 Terrak and coworkers established a TGase assay based on measuring tryptophan fluorescence changes. 104 They selected S. aureus MgtA as a model enzyme that was devoid of tryptophan residue, 58 and prepared a series of MgtA variants with single tryptophan mutation.…”