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.

Cristina Lento

Department of Chemistry

York University

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

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