Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes

Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins.

show abstract

“… 19 , 28 Lipodisq MS may also assist in the identification of different lipids and/or ligands present in crystal structures. 76 …”

Section: Discussionmentioning

confidence: 99%

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins

et al. 2021

View full text Add to dashboard Cite

show abstract

“…In addition, via molecular dynamics simulations of in silico built models for Cx46/Cx50 heteromers, in this case α type Cxs, results in highly similar interfacial interactions ( 10 ). Further structural studies to confirm the stoichiometry of heteromeric Cx hemichannels might be done via complementary methods such as native MS, as we have already shown on several membrane protein complexes ( 35 , 36 , 37 , 38 , 39 ), although a considerable increase in protein concentration and stability should be required prior to undertake this new task. Nevertheless, that approach is not able to provide the subunit arrangement, which can be effectively stated herein via AFM imaging.…”

Section: Discussionmentioning

confidence: 99%

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging

Naulin

Lozano

Fuentes

et al. 2020

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. While homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy (AFM). For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of 3 Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements which can be derived from the law of total probabilities. AFM imaging of ring-like structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology

show abstract

“…They may serve as mediators in biological processes, including protein-ligand, protein-protein and signalling (Shehu & Kavraki, 2012). The characterisation of protein using wet techniques such as X-ray crystal or NMR is often challenging due to their amphipathic nature and the ligand-binding requirements (Montenegro et al, 2017). During the protein crystallisation, secondary structures such as helix and strand may unfold/ transition directly to the loop due to the hydrogen bonding reduction and other reasons (Debenedictis & Keten, 2019).…”

Section: Comparative Ag85c Protein Loop Dynamics and Distance Matricesmentioning

confidence: 99%

Weak spots inhibition in the Mycobacterium tuberculosis antigen 85C target for antitubercular drug design through selective irreversible covalent inhibitor-SER124

Adewumi

El-Rashedy

Soremekun

et al. 2020

Journal of Biomolecular Structure and Dynamics

View full text Add to dashboard Cite

Mycobacterium tuberculosis (Mtb) encoded secreted antigen 85 enzymes (Ag85A/Ag85B/Ag85C) play that critical roles in the virulence, survival and drug-resistant TB of the pathogen. Ag85 proteins are potential antitubercular drug targets because they are essential in the catalytic synthesis of trehalose moieties and mycolic acid attachment to the Mtb cell wall. Recently, experimental protocols led to the discovery of a selective covalent Ag85 inhibitor, b-isomer monocyclic enolphosphorus Cycliphostin (CyC 8b) compound, which targets the Ag85 serine 124 to exhibit a promising therapeutic activity. For the first time, our study unravelled the structural features among Mtb Ag85C homologs and motions and dynamics of Ag85C when the CyC 8b bound covalently and in open model conformations to the protein using bioinformatics tools and integrated Molecular dynamics simulations. Comparative Ag85C sequence analysis revealed conserved regions; 70% active site, 90% Adeniyi loop L1 and 50% loop L2, which acts as a switch between open and closed conformations. The average C-a atoms RMSD (2.05 Å) and RMSF (0.9 Å) revealed instability and high induced flexibility in the CyC 8b covalent-bound compared to the apo and open model systems, which displayed more stability and lower fluctuations. DSSP showed structural transitions of a-helices to bend and loops to 3 10-helices in the bound systems. SASA of CyC 8b covalent bound showed active site hydrophobic residues exposure to huge solvent. Therefore, these findings present the potential opportunity hotspots in Ag85C protein that would aid the structure-based design of novel chemical entities capable of resulting in potent antitubercular drugs.

show abstract

Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes

Cited by 13 publications

References 59 publications

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins

Detergent-free Lipodisq Nanoparticles Facilitate High-Resolution Mass Spectrometry of Folded Integral Membrane Proteins

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging

Weak spots inhibition in the Mycobacterium tuberculosis antigen 85C target for antitubercular drug design through selective irreversible covalent inhibitor-SER124

Contact Info

Product

Resources

About