Analysis of Membrane Proteins

Therefore, the importance of the conditions under which biological molecules are examined in the infrared needs to be emphasised. It is observed here, using the example of myelin basic protein, that this protein is very sensitive to the surroundings. The lipid environment more closely mimics the native environment, and thus presumably gives a far bettei indication of the native structure. 

As previously mentioned in Section 6.3, infrared spectroscopy can be used to investigate the conformations of lipids. Remember that we looked at the conformation of phosphatidylserine (PS) by using the deconvolved carbonyl band. We will now look at changes to this lipid due to the presence of a protein. 

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Chevallet, M. Santoni, V. Poinas, A. Rouquie, D. Fuchs, A. Kieffer, S. Rossignol, M. Lunardi, J. Garin, J. Rabilloud, T. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 1998,19,1901-1909. 

Lohaus, C., Nolte, A., Blueggel, M., Scheer, C., Klose, J., Gobom, J., Schueler, A., Wiebrin-ghaus, T., Meyer, H.E., Marcus, K. (2007). Multidimensional chromatography a powerful tool for the analysis of membrane proteins in mouse brain. J. Proteome Res. 6(1), 105-113. 

Xiang, R., Shi, Y., Dillon, D.A., Negin, B., Horvath, C., Wilkins, J.A. (2004). 2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474. J. Proteome Res. 3, 1278-1283. 

Bill, R.M. (2001) Yeast - a panacea for the structure—function analysis of membrane proteins Current Genetics, 40 (3), 157-171. 

Wang, K., and Richards, F. (1974) An approach to nearest neighbor analysis of membrane proteins. Application to the human erythrocyte membrane of a method employing cleavable cross-linkages. J. Biol. Chem. 249, 8005-8018. 

Olsen JY, Andersen JR, Nielsen PA, Nielsen ML, Figeys D, Mann M, Wisniewski JR. HysTag—a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain. Mol Cell Proteomics 2004 3 82-92. 

At the start of the twenty-first century, the pace of membrane protein structure determinations is clearly accelerating (Figure 1). With the exceptions of rhodopsin (Palczewski et al., 2000) and the calcium ATPase (Toyoshima et al., 2000), however, eukaryotic channels, transporters, and receptors are conspicuously absent from the list of known membrane protein structures. These two exceptions, as proteins of naturally high abundance, highlight the current reality that no structure has been determined for an overexpressed eukaryotic membrane protein. This situation reflects the present difficulties in the reliable overexpression of membrane proteins, particularly those of eukaryotic organisms. Just as the development 20 years ago of overexpression systems for water-soluble proteins revolutionized the structure determinations of this class of proteins, advances in membrane protein expression will be essential to successful realization of the goal of routine structural analysis of membrane proteins. 

Thus, everything concurs in making membrane proteomics probably one of the most difficult sub-field in proteomics. Up to now, the proteomics toolbox that we can use has proven quite imperfect to provide a thorough, quantitative and precise (i.e. including post-translational modifications analysis) analysis of membrane proteins. 

Two-dimensional BAC/SDS polyacrylamide gel electrophoresis has been established as further tool in the field of proteome research, especially regarding the separation and analysis of membrane proteins. It is by far more efficient in resolving membrane proteins than common 2-DE and furthermore can be utilized in a complementary way to one-dimensional SDS-PAGE. Therefore, among other techniques, future proteome studies focussing on membrane proteins should include 2-DB as well. 

Rabilloud, X, Blisnick, T., Heller, M., Luche, S., Aebersold, R., Limardi, J. and Braun-Breton, C. (1999) Analysis of membrane proteins by two-dimensional electrophoresis comparison of the proteins extracted from normal or Plasmodium falciparam-infected erythrocyte ghosts. Electrophoresis 20, 3603-3610. 

Stagljar, I. and Fields, S. (2002) Analysis of membrane protein interactions using yeast-based technologies. Trends. Biochem. Sci. 27, 559-563. 

Pebay-Peyroula E, ed. Biophysical Analysis of Membrane Proteins Investigating Structure and Function. 2008. Wiley-VCH, Weinheim, Germany. 

Werten PJ, Remigy HW, de Groot BL, Fotiadis D, Philippsen A, Stahlberg H, Grubmuller H, Engel A. Progress in the analysis of membrane protein strucmre and function. FEES Letts. 2002 529 65-72. 

J Deisenhofer, O Epp, K Miki, R Huber and H Michel (1984) X-ray structural analysis of membrane protein complex Electron density map at 3 resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. J Mol Biol 180 385-398... 

We beheve that this approach of handhng membrane proteins on solid supports constitutes a promising route for analytical strategies aimed at analysis of membrane proteins and consequently also for the QCM-D technique in the development of patterned surfaces for array-based analysis of biorecognition events. 

Much of what is known about these protein-lipid interactions has come from protein purification and reconstitution of function dependent on lipids. Genetic approaches coupled with in vitro verification of function have uncovered new roles for lipids. Most exciting have been results from X-ray crystallographic analysis of membrane proteins, which have revealed lipids in specific and tight association with proteins. The predominant structural motif for the membrane-spanning domain of membrane proteins is an a-helix of 20-25 amino acids, which is sufficient to span the 30-A core of the bilayer. A 3-barrel motif is also found to a lesser extent. 

Proteomics based on the 2-DE approach, of course, suffers from problems associated with the analysis of membrane proteins, so that such mitochondrial proteins are poorly represented on 2-D profiles. In addition, many mitochondrial proteins are more basic than cytosolic proteins, mitochondria are rich in low molecular weight (<10 kDa) proteins, and mitochondrial proteins are poorly described in databases (Lescuyer et al.,... 

Maslennikov I, Kefala G, Johnson C, Riek R, Choe S, Kwiatkowski W (2007) NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes. BMC Stmct Biol 7 74... 

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