Single-Molecule Spectroscopic Tools for Measuring Microsecond to Millisecond Dynamics of Calmodulin

Abstract

Single-molecule spectroscopy has developed into a powerful tool for the study of biological systems. The ability to observe single protein changes has revealed a great deal of information about the heterogeneity of these systems. In this dissertation, single-molecule techniques have been used to investigate the effect of Ca2+ on millisecond and microsecond fluctuations of the protein calmodulin (CaM). The first part of this dissertation discusses the development and testing of a home-built, two-channel, confocal microscope system used for fluorescence correlation spectroscopy (FCS) and scanning single-molecule measurements. Secondly, the newly built system was tested by performing two-channel FCS measurements using a FRET-pair labeled synthetic polyproline peptide. Polyproline has been shown to approximate a "rigid-rod" and therefore was not expected to show any FRET fluctuations on the FCS timescale. The results from the polyproline correlations led to an investigation to develop expressions to describe the differences in the initial amplitudes of the correlations. These expressions were dependent on the presence of multiple FRET states in the solution and fits were demonstrated using both simulations and real data. Next, the dynamics of several FRET-pair labeled mutants of CaM were measured using FCS techniques. The resulting correlations were globally fit to reveal inter-lobe dynamics on the 100 microsecond timescale that were diminished upon the removal of Ca2+. Intra-lobe dynamics of the N-terminus were also investigated demonstrating an increase in the dynamics in the apo state when compared to the Ca2+ bound state. Finally, CaM was immobilized in unilamellar vesicles to probe millisecond dynamics of the CaM 34-110 mutant in the presence and absence of Ca2+. Rates of interchange between conformational substates of CaM were measured demonstrating an increase in the rates of interchange between conformations in the presence of Ca2+. This supports the view that when bound to Ca2+, CaM is in a more dynamic state leading to its ability to bind a wide variety of targets.