SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis

Naomi L. Pollock, Megha Rai, Kailene S. Simon, Sophie J. Hesketh, Alvin C.k. Teo, Mayuriben Parmar, Pooja Sridhar, Richard Collins, Sarah C. Lee, Zoe N. Stroud, Saskia E. Bakker, Stephen P. Muench, C. Howard Barton, Gregory Hurlbut, David I. Roper, Corinne J.i. Smith, Timothy J. Knowles, Corinne M. Spickett, J. Malcolm East, Vincent PostisTim R. Dafforn

Research output: Contribution to journalArticle

Abstract

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.
Original languageEnglish
Pages (from-to)1437-1445
Number of pages9
JournalBBA -Biomembranes
Volume1861
Issue number8
Early online date28 May 2019
DOIs
Publication statusPublished - 1 Aug 2019

Fingerprint

Styrene
Electrophoresis
Encapsulation
Membrane Proteins
Gels
Lipids
Quaternary Protein Structure
Proteins
Immunoblotting
Cytosol
Electron microscopy
Mass spectrometry
Mass Spectrometry
Phospholipids
Electron Microscopy
maleic acid

Bibliographical note

© 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Funding: European Union under a Marie Skłodwska-Curie Initial Training Network FP7-PEOPLE-2012-ITN Grant Agreement Number 316630 CAS IDP, BBSRC Tools and Resources Development Fund grant number BB/P027482/1.

Keywords

  • Membrane protein
  • Nanoparticle
  • Native PAGE
  • Protein complex
  • SMALP

Cite this

Pollock, N. L., Rai, M., Simon, K. S., Hesketh, S. J., Teo, A. C. K., Parmar, M., ... Dafforn, T. R. (2019). SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis. BBA -Biomembranes, 1861(8), 1437-1445. https://doi.org/10.1016/j.bbamem.2019.05.011
Pollock, Naomi L. ; Rai, Megha ; Simon, Kailene S. ; Hesketh, Sophie J. ; Teo, Alvin C.k. ; Parmar, Mayuriben ; Sridhar, Pooja ; Collins, Richard ; Lee, Sarah C. ; Stroud, Zoe N. ; Bakker, Saskia E. ; Muench, Stephen P. ; Barton, C. Howard ; Hurlbut, Gregory ; Roper, David I. ; Smith, Corinne J.i. ; Knowles, Timothy J. ; Spickett, Corinne M. ; East, J. Malcolm ; Postis, Vincent ; Dafforn, Tim R. / SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis. In: BBA -Biomembranes. 2019 ; Vol. 1861, No. 8. pp. 1437-1445.
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Pollock, NL, Rai, M, Simon, KS, Hesketh, SJ, Teo, ACK, Parmar, M, Sridhar, P, Collins, R, Lee, SC, Stroud, ZN, Bakker, SE, Muench, SP, Barton, CH, Hurlbut, G, Roper, DI, Smith, CJI, Knowles, TJ, Spickett, CM, East, JM, Postis, V & Dafforn, TR 2019, 'SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis', BBA -Biomembranes, vol. 1861, no. 8, pp. 1437-1445. https://doi.org/10.1016/j.bbamem.2019.05.011

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis. / Pollock, Naomi L.; Rai, Megha; Simon, Kailene S.; Hesketh, Sophie J.; Teo, Alvin C.k.; Parmar, Mayuriben; Sridhar, Pooja; Collins, Richard; Lee, Sarah C.; Stroud, Zoe N.; Bakker, Saskia E.; Muench, Stephen P.; Barton, C. Howard; Hurlbut, Gregory; Roper, David I.; Smith, Corinne J.i.; Knowles, Timothy J.; Spickett, Corinne M.; East, J. Malcolm; Postis, Vincent; Dafforn, Tim R.

In: BBA -Biomembranes, Vol. 1861, No. 8, 01.08.2019, p. 1437-1445.

Research output: Contribution to journalArticle

TY - JOUR

T1 - SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis

AU - Pollock, Naomi L.

AU - Rai, Megha

AU - Simon, Kailene S.

AU - Hesketh, Sophie J.

AU - Teo, Alvin C.k.

AU - Parmar, Mayuriben

AU - Sridhar, Pooja

AU - Collins, Richard

AU - Lee, Sarah C.

AU - Stroud, Zoe N.

AU - Bakker, Saskia E.

AU - Muench, Stephen P.

AU - Barton, C. Howard

AU - Hurlbut, Gregory

AU - Roper, David I.

AU - Smith, Corinne J.i.

AU - Knowles, Timothy J.

AU - Spickett, Corinne M.

AU - East, J. Malcolm

AU - Postis, Vincent

AU - Dafforn, Tim R.

N1 - © 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ Funding: European Union under a Marie Skłodwska-Curie Initial Training Network FP7-PEOPLE-2012-ITN Grant Agreement Number 316630 CAS IDP, BBSRC Tools and Resources Development Fund grant number BB/P027482/1.

PY - 2019/8/1

Y1 - 2019/8/1

N2 - Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.

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KW - Membrane protein

KW - Nanoparticle

KW - Native PAGE

KW - Protein complex

KW - SMALP

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