Submicro to Millisecond Conformational Transitions of Bacteriophage T4 Lysozyme with Ångström Accuracy using Multiparameter Fluorescence Detection

Abstract

We use single molecule FRET and the completeness of Multiparameter Fluorescence Detection to study protein flexibility and dye rigidity from submicro- to millisecond timescales in native and non-native conditions of the bacteriophage T4 Lysozyme (T4L). T4L contains 164-residues and consists of two domains connected by a long alpha helix. The enzyme cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of bacterial cell wall saccharides. From crystalographic and EPR measurement the native enzyme is believed to be in dynamic exchange between various “open” conformations. Most likely a set of hinges is responsible for the dynamic motion of the enzyme. However, the time scale of these hinges is still unknown. To accurately determine protein conformations from single molecule FRET, one needs to consider the dynamic behavior of donor and acceptor fluorophores. To test protein and dye mobility, we used single and double labeled T4L mutants with various fluorophore linkers using site specific mutants. The orthogonal system contains a ketone handle in the N-terminal domain for reaction with a hydroxylamine or hydrazide fluorophore, or a thiol group in the C-terminal domain for reaction with a thiol-specific fluorophore. Furthermore, the double mutant is ideal for site-specific attachment of the donor and acceptor fluorophores. Beside studies on native conformational dynamics we look into non native conformational transitions using chemical denaturants. In addition, we complement our experimental work by modelling the accesible volume (AV) required for each fluorophore using steric interaction with known crystalographic data. The AV model considers all available locations where the dye could be located without causing sterical clashes. This tool in combination with single molecule FRET allows us to quantitatively determine corresponding structural distances from experimental data.