Dataset Information

A general approach to visualize protein binding and DNA conformation without protein labelling.

ABSTRACT: Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.

SUBMITTER: Song D

PROVIDER: S-EPMC4786781 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A general approach to visualize protein binding and DNA conformation without protein labelling.

Song Dan D Graham Thomas G W TG Loparo Joseph J JJ

Nature communications 20160308

Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in ...[more]

PMID: 26952553

Similar Datasets

Project description:Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

Dataset Information

A general approach to visualize protein binding and DNA conformation without protein labelling.

Publications

A general approach to visualize protein binding and DNA conformation without protein labelling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets