HPLC‐SEC Characterization of Membrane Protein‐Detergent Complexes

Alla Korepanova1, Edmund D. Matayoshi2

1 Department of Biochemistry, Advanced Technologies, Abbott Laboratories, Abbott Park, Illinois, 2 Department of Structural Biology, Advanced Technologies, Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.5
DOI:  10.1002/0471140864.ps2905s68
Online Posting Date:  April, 2012
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Abstract

Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent‐solubilized membrane proteins. However, size‐exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein‐detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high‐performance liquid chromatography SEC and SLS‐UV‐RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein‐detergent complexes. Curr. Protoc. Protein Sci. 68:29.5.1‐29.5.12. © 2012 by John Wiley & Sons, Inc.

Keywords: membrane protein; protein‐detergent complexes; static light; scattering; size‐exclusion chromatography; HPLC‐SEC

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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Literature Cited

Literature Cited
   Folta‐Stogniew, E. 2006. Oligomeric states of proteins determined by size‐exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol. Biol. 328:97‐112.
   Folta‐Stogniew, E. and Williams, K. 1999. Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J. Biomol. Tech. 10:51‐63.
   Hayashi, Y., Matsui, H., and Takagi, T. 1989. Membrane protein molecular weight determined by low‐angle laser light‐scattering photometry coupled with high‐performance gel chromatography. Methods Enzymol. 172:514‐528.
   Kendrick, B.S., Kerwin, B.A., Chang, B.S., and Philo, J.S. 2001. Online size‐exclusion high‐performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein‐protein or protein‐ligand association states. Anal. Biochem. 299:136‐146.
   Kunji, E.R., Harding, M., Butler, P.J., and Akamine, P. 2008. Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography. Methods 46:62‐72.
   Moller, J.V. and le Maire, M. 1993. Detergent binding as a measure of hydrophobic surface area of integral membrane proteins. J. Biol. Chem. 268:18659‐18672.
   Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411‐2423.
   Ravaud, S., Do Cao, M.A., Jidenko, M., Ebel, C., Le Maire, M., Jault, J.M., Di Pietro, A., Haser, R., and Aghajari, N. 2006. The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent‐solubilized state. Biochem. J. 395:345‐353.
   Slotboom, D.J., Duurkens, R.H., Olieman, K., and Erkens, G.B. 2008. Static light scattering to characterize membrane proteins in detergent solution. Methods 46:73‐82.
   Strop, P., and Brunger, A.T. 2005. Refractive index‐based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14:2207‐2211.
   Wen, J., Arakawa, T., and Philo, J.S. 1996. Size‐exclusion chromatography with on‐line light‐scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal. Biochem. 240:155‐166.
   Winstone, T.L., Jidenko, M., le Maire, M., Ebel, C., Duncalf, K.A., and Turner, R.J. 2005. Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand. Biochem. Biophys. Res. Commun. 327:437‐445.
   Wisniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. 2009. Universal sample preparation method for proteome analysis. Nat. Methods 6:359‐362.
   Wyatt, P.J. 1991. Combined differential light scattering with various liquid chromatography separation techniques. Biochem. Soc. Trans. 19:485.
   Wyatt, P. 1993. Light scattering and the absolute characterization of macromolecules. Anal. Chim. Acta 272:1‐40.
   Yernool, D., Boudker, O., Folta‐Stogniew, E., and Gouaux, E. 2003. Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42:12981‐12988.
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