Matthew S. DeVore

Detecting intramolecular dynamics and multiple Förster resonance energy transfer states by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.

Single-Molecule FRET States, Conformational Interchange, and Conformational Selection by Dye Labels in Calmodulin

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 μs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca(2+), the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging.

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.

Three dimensional time-gated tracking of non-blinking quantum dots in live cells

Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.

Investigations of Calmodulin Conformations Resolved by Single Molecule Microscopy

Measurements of the distance between two dye molecules covalently linked to the calcium signaling protein calmodulin (CaM) have been previously performed by our group to investigate the conformations of CaM in solution. It was shown that calmodulin exists in a wide range of distinct conformations whose amplitudes depend upon free calcium concentrations (1). Currently, we are investigating affects that the choice of dye pair or labeling site has on measured conformations. This is done using an alternating laser excitation (ALEX) single molecule microscope system that has been custom built in our laboratory. Time correlated single photon counting in bulk samples is used to determine the time resolved anisotropy of the dye pair and the orientational mobility of each dye. Analysis of burst measurements using interphoton time burst selection criteria and the probability distribution analysis reveal a wide range of CaM conformations. Conformational analysis is performed using both discrete states and the maximum entropy method. The maximum entropy method reveals the most probable underlying conformational distribution that fits our data. Finally, we are investigating fluorescence fluctuations within CaM conformations using conformationally sorted fluorescence correlation spectroscopy.1. Slaughter et al., J. Phys. Chem. B, 2004, 108, 10388-10397