Dataset Information

Carbene Footprinting Accurately Maps Binding Sites in Protein-Ligand and Protein-Protein Interactions

ABSTRACT: Specific interactions between proteins and their binding partners are fundamental to life processes. Differential protein footprinting using a new and efficient, photo-activated aryltrifluromethylcarbene probe together with mass spectrometry has been employed to identify protein-ligand and protein-protein interaction sites. In a model protein-small molecule system, the location of a penta-N-acetylchitopentaose carbohydrate substrate was accurately mapped to the binding cleft of lysozyme. As a fine detail, footprinting revealed disruption of an intramolecular hydrogen-bond network within lysozyme, which is known to be associated with substrate binding. In a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5, and a diubiquitin substrate were located to different functional domains thus clarifying uncertainties from an X-ray crystal structure of USP5. The much improved properties of this bespoke diazirine probe make differential carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human) Gallus Gallus (chicken)

SUBMITTER: Lucio Manzi

LAB HEAD: Neil J. Oldham

PROVIDER: PXD004971 | Pride | 2016-12-19

REPOSITORIES: Pride

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Publications

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.

Manzi Lucio L Barrow Andrew S AS Scott Daniel D Layfield Robert R Wright Timothy G TG Moses John E JE Oldham Neil J NJ

Nature communications 20161116

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficie ...[more]

PMID: 27848959

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Project description:To define the ECF sigma sigV - regulated genes during log growth phase in LB media under induction conditions for sigV The seven extracytoplasmic function (ECF) sigma (σ) factors of Bacillus subtilis are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σV as including at least 20 operons many of which are also regulated by σM, σX, or σW. The σV regulon is strongly and specifically induced by lysozyme and this induction is key to the intrinsic resistance of B. subtilis to lysozyme. Strains with null mutations in either sigV or in all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σV in the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX or σW does not. Lysozyme resistance results from the ability of σV to activate the transcription of two operons: the autoregulated sigV-rsiV-oatA-yrhK operon and dltABCDE. Genetic analyses reveal that oatA and dlt are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity whereas a double oatA dltA mutant is as sensitive as a sigV null strain. Moreover, the triple sigV oatA dltA mutant is no more sensitive than the oatA dltA double mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, σV confers lysozyme resistance by activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and D-alanylation of teichoic acids by DltABCDE. Strains Δ7Pxyl-sigV + xylose vs. Δ7Pxyl-sigV - xylose, 168 + lysozyme vs. 168 - lysozyme. Each experiment was conducted 6 times using three independent total RNA preparations (biologlical triplicates). For each paired comparison, one sample was labeled with Alexa Fluor 555 and the other was with Alexa Fluor 647. For each comparison, three replicates were performed with dyeswap with the same RNA preparation.

Dataset Information

Carbene Footprinting Accurately Maps Binding Sites in Protein-Ligand and Protein-Protein Interactions

Publications

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.

Similar Datasets

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