FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins

Watkinson, Thomas G.; Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.

doi:10.1007/s13361-016-1421-1

Cited by 34 publications

(33 citation statements)

References 39 publications

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“…Figure is reproduced from Ref. [218]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)…”

Section: Other Chemical Labelling Methodsmentioning

confidence: 99%

“…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]. This study revealed additional intermolecular contacts with the amphipol (Fig.…”

Section: Hydroxyl Radical Footprintingmentioning

confidence: 99%

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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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“…Figure is reproduced from Ref. [218]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)…”

Section: Other Chemical Labelling Methodsmentioning

confidence: 99%

Section: Hydroxyl Radical Footprintingmentioning

confidence: 99%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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“…Residue-level resolution can also be achieved by proteolytic digestion followed by liquid chromatography LC-MS/MS analysis (i.e., bottom-up approach). To date, protein footprinting has been employed to research the HOS of a plethora of large systems such as antibodies, large multi-protein assemblies, viruses, membrane proteins embedded in micelles, nanodiscs, and intact cells (Baerga-Ortiz et al, 2002;Lanman et al, 2004;Guan & Chance, 2005;Coales et al, 2009;Houde et al, 2009;Espino, Mali, & Jones, 2015;Lu et al, 2016;Watkinson et al, 2017;Zhu et al, 2017). In this review, we will look at the contributions of Dr. Michael Gross to structural biology, specifically, in the field of protein footprinting.…”

Section: Introductionmentioning

confidence: 99%

The Making of a Footprint in Protein Footprinting: A Review in Honor of Michael L. Gross

McKenzie‐Coe

Shortt

Jones

2020

Mass Spectrometry Reviews

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Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprintingbased approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein-protein interactions, and protein-ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure.

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“…One report suggested that only sulfur-containing amino acids, which are highly reactive, yield modified amino acids using FPOP [13]. Nanodiscs and detergents are commonly used to increase the hydroxyl radical labeling during the membrane protein FPOP studies [14,15]. Yet, it was hypothesized that the membrane environment scavenged the hydroxyl radicals during FPOP, compromising the FPOP analysis.…”

Section: Introductionmentioning

confidence: 99%

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

Cheng

Mobley²,

Misra³

et al. 2020

Preprint

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The analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. Here, we investigated the effect of bulk hydroxyl radical scavenging in FPOP using both isolated cellular membranes as well as detergent micelles. We found no significant change in radical scavenging activity upon the addition of disrupted cellular membranes with the membrane concentration in the range of 0-25600 cell/μL using an inline radical dosimeter. We confirmed the non-scavenging nature of the membrane with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration acts as a potent hydroxyl radical scavenger as expected, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. These results suggest that any scavenging effect of membranes or organized amphiphilic membrane mimetics in FPOP experiments are not due to bulk hydroxyl radical scavenging, but may be due to a localized scavenging phenomenon.

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FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins

Cited by 34 publications

References 39 publications

Mass spectrometry-enabled structural biology of membrane proteins

Mass spectrometry-enabled structural biology of membrane proteins

The Making of a Footprint in Protein Footprinting: A Review in Honor of Michael L. Gross

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

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