Towards integrative structural mass spectrometry: Benefits from hybrid approaches

Marcoux, Julien; Cianférani, Sarah

doi:10.1016/j.ymeth.2015.05.024

Cited by 25 publications

(23 citation statements)

References 163 publications

(185 reference statements)

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“…5). In contrast to traditional bottom-up proteomics relying on trypsin digestion, the top-down MS approach consists of the analysis of entire proteins, enabling the identification of combinations of PTMs and the direct semiquantification of proteoforms (23)(24)(25). This approach is particularly relevant for the analysis of C. glutamicum porins that are rather hydrophobic with the presence of PTMs consisting of C32-C36 mycoloyl residues and that lack arginine and lysine residues for the generation of adequate tryptic peptides.…”

Section: Discussionmentioning

confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.Corynebacteriales | O-acylation | sequence motif | top-down proteomics | NMR

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Section: Discussionmentioning

confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…( [5][6][7][8] Hydrogen/Deuterium exchange detected by mass spectrometry (HDX-MS) is a methodology applied to proteins in recent years for investigating their properties, at peptide resolution, such as dynamics, conformational changes, post-translational modifications, viral capsid assembly, spontaneous and assisted folding, and interactions with partners or ligands. (9)(10)(11)(12)(13) The method is , that it has great resolution and sensitivity, that it allows for an accessible mass range of up to 10 6 Da, that it allows high speed analysis and that it is a nonensemble technique; each single spectrum contains extractible information about all discrete coexisting states of the protein of interest.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

2017

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Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

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“…The examples of the work on OXPHOS complexes permit an illustration of the promises that new proteomics-based methods offer, but also of their limitations, as to invite their critical implementation in the investigations on bacterial systems. For the interested reader who likes to learn more about new developments and their applications, we would like to refer to a number of recent reviews (Boersema, Kahraman, & Picotti, 2015;Catherman et al, 2014;Cui, Rohrs, & Gross, 2011;Hyung & Ruotolo, 2012;Marcoux & Cianf erani, 2015;Owens, 2011;Petrotchenko & Borchers, 2014;Zhou & Robinson, 2010).…”

Section: Respiratory Protein Complexes In Assemblymentioning

confidence: 99%

Bacterial Electron Transfer Chains Primed by Proteomics

Wessels¹,

Almeida²,

Kartal³

et al. 2016

Advances in Bacterial Electron Transport Systems and Their Regulation

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Electron transport phosphorylation is the central mechanism for most prokaryotic species to harvest energy released in the respiration of their substrates as ATP. Microorganisms have evolved incredible variations on this principle, most of these we perhaps do not know, considering that only a fraction of the microbial richness is known. Besides these variations, microbial species may show substantial versatility in using respiratory systems. In connection herewith, regulatory mechanisms control the expression of these respiratory enzyme systems and their assembly at the translational and posttranslational levels, to optimally accommodate changes in the supply of their energy substrates. Here, we present an overview of methods and techniques from the field of proteomics to explore bacterial electron transfer chains and their regulation at levels ranging from the whole organism down to the Ångstrom scales of protein structures. From the survey of the literature on this subject, it is concluded that proteomics, indeed, has substantially contributed to our comprehending of bacterial respiratory mechanisms, often in elegant combinations with genetic and biochemical approaches. However, we also note that advanced proteomics offers a wealth of opportunities, which have not been exploited at all, or at best underexploited in hypothesis-driving and hypothesis-driven research on bacterial bioenergetics. Examples obtained from the related area of mitochondrial oxidative phosphorylation research, where the application of advanced proteomics is more common, may illustrate these opportunities.

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Towards integrative structural mass spectrometry: Benefits from hybrid approaches

Cited by 25 publications

References 163 publications

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

Bacterial Electron Transfer Chains Primed by Proteomics

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