Probing the structure of nanodiscs using surface-induced dissociation mass spectrometry

Harvey, Sophie R.; VanAernum, Zachary; Kostelic, Marius; Marty, Michael T.; Wysocki, Vicki H.

doi:10.1039/d0cc05531j

Cited by 17 publications

(27 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All native MS experiments were conducted using a Q Exactive™ Ultra-High Mass Range (UHMR) Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Scientific) that was modified with a customized device for performing surface-induced dissociation ( 66 , 67 ). Samples were diluted to 1 μM in 800 mM NH 4 OAc and transferred to filament-containing borosilicate glass capillary tips (Sutter Instrument, Novato, CA) that were individually pulled in-house using a P-97 micropipette puller (Sutter Instrument).…”

Section: Methodsmentioning

confidence: 99%

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Marathe

Lai

Zahurancik

et al. 2021

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

show abstract

Section: Methodsmentioning

confidence: 99%

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Marathe

Lai

Zahurancik

et al. 2021

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Like POPC/SOPE and DPPC/SOPE nanodiscs, at higher collision voltages, SOPE is preferentially ejected from DPPG/SOPE nanodiscs in positive ionization mode. In contrast to pure PG nanodiscs that split in half at higher collision voltages in positive mode, 24,42 DPPG/SOPE nanodiscs continually lose lipids that carry charge as the collision voltage increases but retain two MSP belts.…”

Section: Resultsmentioning

confidence: 94%

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Kostelic¹,

Zak²,

Jayasekera³

et al. 2021

Preprint

View full text Add to dashboard Cite

Native mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes.

show abstract

“…The membrane protein embedded into nanodiscs are then separated from empty nanodiscs using affinity chromatography and/or size exclusion chromatography. The object comprising the membrane protein of interest is in reality quite heterogeneous, containing a mixture of large and small nanodiscs, with more or less lipids embarked,and varying in all dimensions according to the type of MSP used and the ratio lipid:MSP, etc... [33][34][35][36]. In some cases, contacts are made between the protein and the MSP that stabilize it and makes the MSP visible in 3D reconstruction, as exemplified recently [37,38].…”

Section: Discussionmentioning

confidence: 99%

CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer

Zampieri

Gobet

Robert

et al. 2021

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

Probing the structure of nanodiscs using surface-induced dissociation mass spectrometry

Abstract: In the study of membrane proteins and antimicrobial peptides, nanodiscs have emerged as a valuable membrane mimetic to solubilze these molecules in a lipid bilayer. We present the structural characterization...

Cited by 17 publications

References 33 publications

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer

Contact Info

Product

Resources

About