The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H], cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.

We present here a software suite (ORIGAMI) that facilitates the rapid acquisition and analysis of ion mobility data following collisional activation. ORIGAMI was developed for use on Waters Synapt instruments where data acquisition is achieved by interfacing WREnS (Waters Research Enabled Software) and MassLynx. Two components are presented, the first is ORIGAMI MS which enables activation of ions by sequential increase of collision voltages prior to ion mobility analysis. We demonstrate the use of ORIGAMI on the tetrameric assemblies formed by the proteins concanavalin A (103 kDa) and alcohol dehydrogenase (143 kDa). Activation is performed in the trap collision cell of the Synapt TriWave assembly, where the collision voltage can be ramped from 0-200 V. All of the acquired data is recorded in a single file which simplifies data acquisition. This substantially decreases the time needed to perform a typical activated IM-MS experiment on a single protein charge state from approx. 2 hours to ~25 minutes. Following data acquisition the data is analysed in the second component, ORIGAMI ANALYSE , which allows the user to visualise the effect of activation on the mobility of the parent ion, as well as on any produced fragment ion. The user can export the data in the form of heat maps, waterfall or wire plots. In addition, tools implemented in ORIGAMI enable easy data extraction from single or multiple MassLynx .raw files, in-depth interrogation of large datasets, .

We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H] form of the peptide melittin. Each mobility selected form has a different "fingerprint" dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

Infection by soil transmitted parasitic helminths, such as Trichuris spp , are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)−13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

No abstract