conjugative-mating-assays-for-sequence-specific-analysis-transfer

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

time-resolved-electrospray-ionization-hydrogen-deuterium-exchange

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

York University

Conjugative DNA transfer by the F-plasmid is achieved through a type IV secretion system (T4SS) encoded within the plasmid's transfer region; TraF is one of several F-T4SS proteins essential for F-pilus assembly. In order to identify regions of the protein important for TraF function, a series of deletion mutants were assessed for their ability to recover conjugative transfer in a traF knockout. Interestingly, modification of any region of TraF abolishes pilus synthesis, resulting in a loss of rescue of conjugative function. Dynamic analysis of TraF by time-resolved hydrogen-deuterium exchange revealed that the C-terminal region containing the predicted thioredoxin-like domain is quite structured, while the N-terminal region, predicted to interact with TraH in the intact F-T4SS, was more dynamic.

HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid.

Truncated pilin monomers from Pseudomonas aeruginosa strain K122-4 (ΔK122) have been shown to enter a monomer-dimer equilibrium in solution prior to oligomerization into protein nanotubes. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved hydrogen-deuterium exchange mass spectrometry. Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state.

Dimerization of the type IV pilin from Pseudomonas aeruginosa strain K122-4 results in increased helix stability as measured by time-resolved hydrogen-deuterium exchange.

Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins.

ArticleTotal : 2	Year
Publication title Cited by 2	2017
Publication title Cited by 15	2017

Article	Year
HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid. FEBS letters\| PubMed ID: 26785931	2016
Dimerization of the type IV pilin from Pseudomonas aeruginosa strain K122-4 results in increased helix stability as measured by time-resolved hydrogen-deuterium exchange. Structural dynamics (Melville, N.Y.)\| PubMed ID: 26798830	2016
Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development - A review. Analytica chimica acta\| PubMed ID: 27662755	2016

Cristina Lento

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