title: Unravelling the Intricacy of the Crowded Environment through Tryptophan Quenching in Lysozyme.
abstract: Global conformational modulation of proteins in the presence of crowding agents is well-known. In this work, using the intrinsic tryptophan (Trp) residues of lysozyme, we have studied the effect of the crowding agents on the local conformation of this biomolecule. In presence of the macromolecular crowder Dextran 6, considerable quenching of tryptophan fluorescence was observed, which we have attributed to the enhanced proximity of the surrounding charged residues arising from local perturbation of protein structure. Accessibility of the Trp residues in the presence of crowders as a function of thermal and chemical denaturation has also been monitored using the traditional quenchers, acrylamide and iodide. Quenching in the presence of the crowding agents had to be modeled predominantly using the sphere-of-action model, with the sphere-of-volume "V" being postulated to be a signature of the cage-like environments that can exist in the solutions of such polymeric macromolecular crowding agents. Moreover, percolation of the quencher molecules through the entangled crowder systems was observed to be dependent on the micropolarity of the specific crowding agent being studied, with the neutral quencher acrylamide exhibiting maximum quenching in the presence of Dextran 6, while iodide being charged, exhibits higher quenching efficiency when Dextran 70 was the crowder. Additionally, control studies with the free amino acid tryptophan suggest that the variation in quenching so observed is not only due to the changes in the conformation of lysozyme and hence accessibility of the Trp residues but is also dictated by the underlying details and complexity of the crowder solutions to an appreciable extent.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Delhi , Hauz Khas, New Delhi 110016, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Chemistry, Indian Institute of Technology Delhi , Hauz Khas, New Delhi 110016, India.
authors.forename: Pramit K
authors.identifier: 0000-0002-9593-2577
authors.initials: PK
authors.lastname: Chowdhury
mesh_terms: D000818:Animals; D002645:Chickens; D003911:Dextrans / Q000737:chemistry; D004531:Eggs; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D058767:Protein Unfolding; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003911:Dextrans; D014364:Tryptophan; D009113:Muramidase