Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

Pan, Yan; Brown, Leonid S.; Konermann, Lars

doi:10.1016/j.jmb.2011.04.074

Cited by 37 publications

(51 citation statements)

References 92 publications

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“…Its suitability for soluble and membrane proteins is well established. [67][68][69][70][71][72][73][74] Solvent-accessible Cys and Met side chains are most susceptible to oxidation. Aromatic residues can also exhibit high reactivities, whereas the reaction rates of other amino acids are lower.…”

Section: Introductionmentioning

confidence: 99%

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

Pan

Piyadasa

O'Neil

et al. 2012

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

Pan

Piyadasa

O'Neil

et al. 2012

Journal of Molecular Biology

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“…For IMPs it is not uncommon to observe hydroxyl radical-induced modifications almost exclusively at these sulfur-containing residues [33,54,55]. Our laboratory has recently applied laser-induced oxidative labeling for mapping conformational transitions of bacteriorhodopsin, an IMP with a well known threedimensional structure [42]. In the current work, we extend this approach to probe the topology of an IMP for which no highresolution structural information is available.…”

mentioning

confidence: 99%

“…The location(s) and the extent of labeling can be determined by LC-MS/MS peptide mapping [31,32]. Such experiments have been used to explore conformational features and interactions of IMPs [32][33][34][35][36][37][38][39][40][41][42]. For IMP topology studies, however, this approach remains underutilized.…”

mentioning

confidence: 99%

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Pan

Ruan

Valvano

et al. 2012

J. Am. Soc. Mass Spectrom.

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Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ⋅OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424. Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ⋅OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

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“…Thus, online mixing can allow conformational dynamics in response to ligand binding or another stimulus to be probed on stopped-flow (ms) timescales [252,253]. Such methods have enabled MP folding to be monitored [215]. These fast reacting functional groups have recently been extended further to include diazirines which are also activated by laser irradiation [230,231].…”

Section: Discussionmentioning

confidence: 99%

“…Coupling this fluidics setup with rapid online mixing has allowed the folding mechanisms of proteins to be studied (by initiating folding on-line and irradiating at defined time-points thereafter). In the case of MPs, this has allowed the kinetic folding mechanism of bacteriorhodopsin to be studied [215], and topology mapping/validation of MPs [216,217]. A comparative study also investigated the difference in the solvent accessibility of the outer membrane protein OmpT in amphipol A8-35 compared with the detergent DDM [218].…”

Section: Hydroxyl Radical Footprintingmentioning

confidence: 99%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

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Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

Cited by 37 publications

References 92 publications

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Mass spectrometry-enabled structural biology of membrane proteins

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