Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET.

Background: The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore.

Dynamics of nucleosome invasion by DNA binding proteins.

Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured.

FRET fluctuation spectroscopy of diffusing biopolymers: contributions of conformational dynamics and translational diffusion.

The use of fluorescence correlation spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in fluorescence resonance energy transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments.

Photophysics of backbone fluorescent DNA modifications: reducing uncertainties in FRET.

We have investigated the photophysical properties of backbone fluorescent DNA modifications with the goal of reducing many of the sources of uncertainty commonly encountered in Forster resonance energy transfer (FRET) measurements. We show that backbone modifications constrain rotational motions, providing a way by which the orientation of the dye can be controlled in a predictable manner, and reduce the uncertainties in donor-acceptor distance associated with the flexible linkers commonly used in conjugate chemistry. Rotational rigidity also prevents undesirable dye-DNA interactions, which have been shown to affect the photophysical properties of the dye.

Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA.

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays.