Hydrophobic Transmembrane Domain Research Articles

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Chimeric nanobody-decorated liposomes by self-assembly.

Liposomes as drug vehicles have advantages, such as payload protection, tunable carrying capacity and improved biodistribution. However, due to the dysfunction of targeting moieties and payload loss during preparation, immunoliposomes have yet to be favoured in commercial manufacturing. Here we report a chemical modification-free biophysical approach for producing immunoliposomes in one step through the self-assembly of a chimeric nanobody (cNB) into liposome bilayers. cNB consists of a nanobody against human epidermal growth factor receptor 2 (HER2), a flexible peptide linker and a hydrophobic single transmembrane domain. We determined that 64% of therapeutic compounds can be encapsulated into 100-nm liposomes, and up to 2,500 cNBs can be anchored on liposomal membranes without steric hindrance under facile conditions. Subsequently, we demonstrate that drug-loaded immunoliposomes increase cytotoxicity on HER2-overexpressing cancer cell lines by 10- to 20-fold, inhibit the growth of xenograft tumours by 3.4-fold and improve survival by more than twofold.

Molecular characterization and ontogenetic expression profiles of LPXRFa and its receptor in Japanese flounder (Paralichthys olivaceus)

Investigations concerning the LPXRFa system are rarely conducted in flatfish species. Here, we first identified and characterized lpxrfa and its cognate receptor lpxrfa-r genes in the Japanese flounder (Paralichthys olivaceus). The coding DNA sequence of lpxrfa was 579 bp in length, wich encoded a 192-aa preprohormone that can produce three mature LPXRFa peptides. The open reading frame (ORF) of lpxrfa-r was 1446 bp in size, and encoded a 481-aa LPXRFa-R protein that encompassed seven hydrophobic transmembrane domains. Subsequently, tissue distribution expression profiles of lpxrfa and lpxrfa-r transcripts were assayed by quantitative real-time PCR. The results indicated that expressions of lpxrfa transcripts were detected at the highest levels in the brain of both females and males, however, lpxrfa-r transcripts were remarkablely expressed in the brain tissue of female fish and in the testis tissue of male fish. Furthermore, transcript levels of lpxrfa and lpxrfa-r genes were investigated during early ontogenetic development, with the maximum expression levels at 30 days post-hatching. Overall, these data contribute to providing preliminary proof for the existence and structure of the LPXRFa system in Japanese flounder, and the study is just the foundation for researching physiological function of LPXRFa system in this species.

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

Exploration of LPS2 agonist binding modes using the combination of a new hydrophobic scaffold and homology modeling

Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.

Rationale design of novel PIEZO1 modulators.

Chemical modulators of mechanosensitive Piezo channels would help better understand physiology and could treat certain diseases. A promising small molecule to expand the currently limited Piezo channel pharmacome is Yoda1, a Piezo1-selective activator identified from blind high-throughput screening. We previously uncovered an allosteric Yoda1 binding pocket in Piezo1, sandwiched between two hydrophobic transmembrane domains called Repeats A and B. Here, we show that crosslinking these Repeats with disulfide bridges inhibits the effects of Yoda1, supporting a mechanism in which Yoda1 acts like a wedge by separating these two domains from each other. Using open and shut Piezo1 structural models and rigorous in silico ligand binding free energy calculations, we next show that Yoda1 interacts with this binding region with a higher affinity for the open state, as expected for an allosteric activator. Our free energy calculations further recapitulate structure activity relationships established for seven Yoda1 analogs. Among 155 purchasable compounds virtually selected against a 3D pharmacophore map of the Yoda1 binding site in the open state, several act as bona fide Piezo1 activators with chemical scaffolds distinct from Yoda1 or other known Piezo1 modulators. This work provides a structural and thermodynamic framework for the activation of Piezo1 by Yoda1 and demonstrates the possibility to computationally exploit this pharmacological region to rationally design novel molecules with potential clinical value.

A robust antibody discovery platform for difficult-to-express G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCR-specific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study.

Signal sequences encode information for protein folding in the endoplasmic reticulum.

One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon. How protein translocation coordinates with chaperone availability in the ER to promote protein folding remains unclear. We find that marginally hydrophobic signal sequences and transmembrane domains cause transient retention at the Sec61 translocon and require the luminal BiP chaperone for efficient protein translocation. Using a substrate-trapping proteomic approach, we identify that nascent proteins bearing marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon. Sec63 is co-translationally recruited to the translocation site and mediates BiP binding to incoming polypeptides. BiP binding not only releases translocationally paused nascent chains but also ensures protein folding in the ER. Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent translocation, but translocated proteins are prone to misfold and aggregate in the ER under limited BiP availability. Thus, the signal sequence-guided protein folding may explain why signal sequences are diverse and use multiple protein translocation pathways.

A fijiviral nonstructural protein triggers cell death in plant and bacterial cells via its transmembrane domain.

Southern rice black-streaked dwarf virus (SRBSDV; Fijivirus, Reoviridae) has become a threat to cereal production in East Asia in recent years. Our previous cytopathologic studies have suggested that SRBSDV induces a process resembling programmed cell death in infected tissues that results in distinctive growth abnormalities. The viral product responsible for the cell death, however, remains unknown. Here P9-2 protein, but not its RNA, was shown to induce cell death in Escherichia coli and plant cells when expressed either locally with a transient expression vector or systemically using a heterologous virus. Both computer prediction and fluorescent assays indicated that the viral nonstructural protein was targeted to the plasma membrane (PM) and further modification of its subcellular localization abolished its ability to induce cell death, indicating that its PM localization was required for the cell death induction. P9-2 was predicted to harbour two transmembrane helices within its central hydrophobic domain. A series of mutation assays further showed that its central transmembrane hydrophobic domain was crucial for cell death induction and that its conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining its ability to induce cell death. Its homologues in other fijiviruses also induced cell death in plant and bacterial cells, implying that the fijiviral nonstructural protein may trigger cell death by targeting conserved cellular factors or via a highly conserved mechanism.

Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei

Octopamine and Tyramine are biogenic amines that have been demonstrated to play an important immunological role in white shrimp, Litopenaeus vannamei. G protein-coupled receptors, known as seven-transmembrane domain receptors, are a variety of neurotransmitter receptors which are sensitive to biogenic amines for initiating the cell signaling pathway. In present study, we cloned and characterized an octopamine/tyramine receptor (LvOA/TA-R) from the hemocytes of L. vannamei, with a 1194 b.p. open reading frame that encodes 398 amino acids. Several bioinformatics analyses indicated that LvOA/TA-R had seven conserved hydrophobic transmembrane domains. The phylogenetic analysis and multiple sequence alignment indicated that LvOA/TA-R was orthologous to the OA/TA receptor of tiger shrimp, P. monodon. LvOA/TA-R was expressed in hemocytes and nervous tissue including circumoesphageal connective tissue and the thoracic and abdominal ganglia. Significant increases in LvOA/TA-R occurred in hemocytes of L. vannamei under Vibrio alginolyticus infection within 30–60 min of infection. Here, we demonstrated that LvOA/TA-R expression is upregulated in response to Vibrio alginolyticus infection and appears to be functionally responsible for the observed immune response. These results suggest that LvOA/TA-R mediates regulation of immunity, which promotes the resistance of L. vannamei to V. alginolyticus.

In Situ Assembly of Transmembrane Proteins from Expressed and Synthetic Components in Giant Unilamellar Vesicles.

Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.

Ribosome-nascent Chain Interaction Regulates N-terminal Protein Modification

Numerous proteins initiate their folding, localization, and modifications early during translation, and emerging data show that the ribosome actively participates in diverse protein biogenesis pathways. Here we show that the ribosome imposes an additional layer of substrate selection during N-terminal methionine excision (NME), an essential protein modification in bacteria. Biochemical analyses show that cotranslational NME is exquisitely sensitive to a hydrophobic signal sequence or transmembrane domain near the N terminus of the nascent polypeptide. The ability of the nascent chain to access the active site of NME enzymes dictates NME efficiency, which is inhibited by confinement of the nascent chain on the ribosome surface and exacerbated by signal recognition particle. In vivo measurements corroborate the inhibition of NME by an N-terminal hydrophobic sequence, suggesting the retention of formylmethionine on a substantial fraction of the secretory and membrane proteome. Our work demonstrates how molecular features of a protein regulate its cotranslational modification and highlights the active participation of the ribosome in protein biogenesis pathways via interactions of the ribosome surface with the nascent protein.

A common evolutionary origin reveals fundamental principles of protein insertases.

Membrane proteins require protein machineries to insert their hydrophobic transmembrane domains (TMDs) into the lipid bilayer. A functional analysis of protein insertases in this issue of PLOS Biology reveals that the fundamental mechanism of membrane protein insertion is universally conserved.

Structure and dynamics of the SARS-CoV-2 envelope protein monomer.

Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

An advanced yeast model to investigate the interplay between structure and function of human gamma-secretase and its substrates

The promiscuous nature of the Alzheimer’s disease associated gamma-secretase (gSec) amongst its substrates remains mysterious after decades of research. In contrast to most other aspartate proteases, gSec cleaves its substrates within the hydrophobic transmembrane domain (TMD), releases an intracellular domain (ICD) and further processes the remaining fragment prior release into the extracellular compartment. Aggregation of the extracellular fragment of the substrate Amyloid Precursor Protein (APP) is related to Alzheimer’s disease.

NMR Detection and Structural Modeling of the Ethylene Receptor LeETR2 from Tomato.

The gaseous plant hormone ethylene influences many physiological processes in plant growth and development. Plant ethylene responses are mediated by a family of ethylene receptors, in which the N-terminal transmembrane domains are responsible for ethylene binding and membrane localization. Until now, little structural information was available on the molecular mechanism of ethylene responses by the transmembrane binding domain of ethylene receptors. Here, we screened different constructs, fusion tags, detergents, and purification methods of the transmembrane sensor domain of ethylene receptors. However, due to their highly hydrophobic transmembrane domain (TMD), only a KSI-fused LeETR21–131 from tomato yielded a good-quality nuclear magnetic resonance (NMR) spectrum in the organic solvent. Interestingly, a dimer model of LeETR21–131 built by the AlphaFold2 algorithm showed greatly converged structures. The interaction analysis of ethylene and LeETR21–131 using molecular docking and molecular dynamics (MD) simulations demonstrated the potential binding sites of ethylene in LeETR21–131. Our exploration provides valuable knowledge for further understanding of the ethylene-perception process in ethylene receptors.

Molecular Modeling Strategies of Cancer Multidrug Resistance.

Cancer remains a leading cause of morbidity and mortality worldwide. Hence, the increase in cancer cases observed in the elderly population, as well as in children and adolescents, makes human malignancies a prime target for anticancer drug development. Although highly effective chemotherapeutic agents are continuously developed and approved for clinical treatment, the major impediment towards curative cancer therapy remains multidrug resistance (MDR). In recent years, intensive studies have been carried out on the identification of new therapeutic molecules to reverse MDR efflux transporters of the ATP-binding cassette (ABC) superfamily. Although a great deal of progress has been made in the development of specific inhibitors for certain MDR efflux pumps in experimental studies, advanced computational studies can accelerate this drug development process. In the literature, there are many experimental studies on the impact of natural products and synthetic small molecules on the reversal of cancer MDR. Molecular modeling methods provide an opportunity to explain the activity of these molecules on the ABC-transporter family with non-covalent interactions as well as it is possible to carry out studies for the discovery of new anticancer drugs specific to MDR with these methods. The coordinate file of the 3-dimensional (3D) structure of the target protein is indispensable for molecular modeling studies. In some cases where a 3D structure cannot be obtained by experimental methods, the homology modeling method can be applied to obtain the file containing the target protein's information including atomic coordinates, secondary structure assignments, and atomic connectivity. Homology modeling studies are of great importance for efflux transporter proteins that still lack 3D structures due to crystallization problems with multiple hydrophobic transmembrane domains. Quantum mechanics, molecular docking and molecular dynamics simulation applications are the most frequently used molecular modeling methods in the literature to investigate non-covalent interactions between the drug-ABC transporter superfamily. The quantitative structure-activity relationship (QSAR) model provides a relationship between the chemical properties of a compound and its biological activity. Determining the pharmacophore region for a new drug molecule by superpositioning a series of molecules according to their physicochemical properties using QSAR models is another method in which molecular modeling is used in computational drug development studies with ABC transporter proteins. There are also in silico absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) studies conducted to make a prediction about the pharmacokinetic properties, and drug-likeness of new molecules. Drug repurposing studies, which have become a trending topic in recent years, involve identifying possible new targets for an already approved drug molecule. There are few studies in the literature in which drug repurposing performed by molecular modelling methods has been applied on ABC transporter proteins. The aim of the current paper is to create a complete review of drug development studies including aforementioned molecular modeling methods carried out between the years 2019-2021. Furthermore, an intensive investigation is also conducted on licensed applications and free web servers used in in silico studies. The current review is an up-to-date guide for researchers who plan to conduct computational studies with MDR transporter proteins.

Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

Diethylpyrocarbonate Footprints a Membrane Protein in Micelles.

Membrane proteins play crucial roles in cell signaling and transport and, thus, are the targets of many small molecule drugs. The characterization of membrane protein structures poses challenges for the high-resolution biophysical tools because the transmembrane (TM) domain is hydrophobic, opening an opportunity for mass spectrometry (MS)-based footprinting. The hydrophobic reagent diethylpyrocarbonate (DEPC), a heavily studied footprinter for water-soluble proteins, can label up to 30% of surface residues via a straightforward protocol, streamlining the MS-based footprinting workflow. To test its applicability to membrane proteins, we footprinted vitamin K epoxide reductase (VKOR) membrane protein with DEPC. The results demonstrate that besides labeling the hydrophilic extracellular (extramembrane (EM)) domain, DEPC can also diffuse into the hydrophobic TM domain and subsequently label that region. The labeling process was facilitated by tip sonication to enhance reagent diffusion into micelles. We then analyzed the correlation between the residue modification extent and the theoretical accessible surface area percentage (%ASA); the data generally show good correlation with the residue location. Compared with conventional hydrophilic footprinters, the relatively hydrophobic DEPC can map a membrane protein's TM domain, suggesting that the reagent's hydrophobicity can be exploited to obtain structural information on the membrane-spanning region. This encouraging result should assist in the development of more efficient footprinters for membrane protein TM domain footprinting, enabled by further understanding the relationship between a reagent's hydrophobicity and its preferred labeling sites.

Carbocation Footprinting of Soluble and Transmembrane Proteins

Here, we introduce carbocations (R3C+) 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.