Conformational dynamics of proteins by fluorescence fluctuation spectroscopy
Yirdaw, Robel Birru
Proteins, as intrinsically flexible molecules, exhibit internal motions at equilibrium. In general, the internal motions consist of changes in the three dimensional coordinates of the constituent atoms. These motions are collectively referred to as conformational dynamics and span multiple orders of magnitude in timescale. Protein conformational dynamics is central to biological processes. In this work, fluorescence fluctuation spectroscopy (FFS), a single molecule technique, was utilized to study the conformational dynamics of Bacteriophage T4 lysozyme (T4L). Bacteriophage T4 is a virus that infects <i>E. coli</i> and T4L is used to break down the <i>E. coli</i> cell wall in the late stages of the infection cycle. The structure of T4L consists of two domains joined by a long helix with the active site situated in between the two domains. The presence of equilibrium conformational dynamics in T4L consisting of the motion of one domain relative to the other has long been postulated. In the work presented here, fluorescence correlation spectroscopy (FCS) and brightness (cumulant) analysis, two complementary variations of FFS, were used to investigate T4 lysozyme conformational dynamics. FCS results give direct evidence for conformational dynamics in T4L consisting of changes in relative distance and orientation of the two domains with a relaxation time of approximately 15 µs. The amplitude of this motion diminishes upon covalent substrate trapping. FCS results indicate the presence of dynamics along the long helix that persists upon substrate binding which may be necessary to facilitate the opening and closing of the active site. Furthermore, in contrast to a motion that involves discrete open and closed conformational states, results point to the presence of multiple conformations. Moreover, FFS was applied on Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CAMKII) and the membrane transporter MsbA. Relative to T4L, these two serve as examples of complex protein systems. The particular challenges in applying FFS to such systems are presented. The use of brightness analysis, a relatively new approach compared to FCS, for the study of protein conformational dynamics is discussed. As part of this work, data analysis and simulation software tools were developed using MATLAB.