Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

Cheng, Ming; Guo, Chunyang; Li, Weikai; Gross, Michael L.

doi:10.1002/anie.202014096

Cited by 11 publications

(19 citation statements)

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“…In a few studies, investigators have applied oxidative methodology or other reactive species such as trifluoromethyl and iodide radical and carbenes to integral membrane proteins. 15,20,28,29 These studies report that significant labeling of amino acid side chains in the extramembrane or solventaccessible regions of the proteins occurred. Recently, the use of aryldiazirine photoactivation to yield a carbene was reported to footprint the hydrophobic transmembrane region on an outer membrane porin protein F. 19 Given the amphipathic nature of the TFB + carbocation as governed by the hydrophobic CF 3 group balanced by the positive charge, we postulated that the TFB + may incorporate in detergent molecules and footprint the hydrophobic transmembrane domain in addition to the extracellular hydrophilic domain.…”

Section: ■ Membrane Protein Footprintingmentioning

confidence: 99%

“…Thus, our ability to footprint membrane protein structure and interactions significantly lags that of their soluble counterparts. In a few studies, investigators have applied oxidative methodology or other reactive species such as trifluoromethyl and iodide radical and carbenes to integral membrane proteins. ,,, These studies report that significant labeling of amino acid side chains in the extramembrane or solvent-accessible regions of the proteins occurred. Recently, the use of aryldiazirine photoactivation to yield a carbene was reported to footprint the hydrophobic transmembrane region on an outer membrane porin protein F …”

Section: Membrane Protein Footprintingmentioning

confidence: 99%

“…Recently, a free radical footprinter, perfluoroisopropyl iodide, was developed that, together with tip sonication to assist partitioning into the membrane, provides 100% footprinting of Y and W for both the extramembrane domain and intramembrane domain of a membrane protein. 20 Exploiting the advantage of tailoring the polarity of the precursor will enable new, highly reactive species and expand the repertoire of protein footprinters.…”

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Carbocation Footprinting of Soluble and Transmembrane Proteins

Gross

2021

Anal. Chem.

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Here, we introduce carbocations (R 3 C + ) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB + ) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB + increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo-and holo-myoglobin footprints shows that the TFB + footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB + is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB + with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra-and trans-membrane domains of membrane proteins.

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Section: ■ Membrane Protein Footprintingmentioning

confidence: 99%

Section: Membrane Protein Footprintingmentioning

confidence: 99%

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confidence: 99%

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Carbocation Footprinting of Soluble and Transmembrane Proteins

Gross

2021

Anal. Chem.

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“…To improve the footprinting performance of the highly hydrophobic reagents on TM domains, a new approach exploiting highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication was developed to ensure efficient transport into the micelle interior (Figure 5), allowing laser dissociation and footprinting of the TM domains [89]. Using the same integral MP, VKOR, as test, we found the PFIPI footprints both the hydrophilic extramembrane and the hydrophobic intramembrane domains.…”

Section: Ms‐based Techniques For Footprinting Mpsmentioning

confidence: 99%

Advances in mass spectrometry‐based footprinting of membrane proteins

Gross

2022

Proteomics

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Structural biology is entering an exciting time where many new high‐resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo‐electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS‐based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

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“…Covalent labeling includes a number of techniques, all of which involve reaction of some reagent with amino acid side chains usually available on the surface of the folded protein. A variety of covalent labeling reagents have been used, including acylation reagents 5 , diethylpyrocarbonate 6 , carbenes 7 , trifluoromethyl radicals 8,9 and iodine radicals 10 . Here, we present an approach based on the use of hydroxyl radicals as a covalent labeling reagent.…”

Section: Introductionmentioning

confidence: 99%

Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

Khaje

Eletsky

Biehn

et al. 2021

Preprint

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High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

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Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

Cited by 11 publications

References 50 publications

Carbocation Footprinting of Soluble and Transmembrane Proteins

Carbocation Footprinting of Soluble and Transmembrane Proteins

Advances in mass spectrometry‐based footprinting of membrane proteins

Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

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