Dataset Information

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

ABSTRACT: Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.

SUBMITTER: Zorrilla S

PROVIDER: S-EPMC2567959 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

Zorrilla Silvia S Ortega Alvaro A Chaix Denis D Alfonso Carlos C Rivas Germán G Aymerich Stéphane S Lillo M Pilar MP Declerck Nathalie N Royer Catherine A CA

Biophysical journal 20080725 9

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control cat ...[more]

PMID: 18658229

Similar Datasets

Project description:The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.

Dataset Information

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

Publications

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets