Methods membrane proteins

In subsequent studies attempting to find a correlation of physicochemical properties and antimicrobial activity, other parameters have been employed, such as Hammett O values, electronic distribution calculated by molecular orbital methods, spectral characteristics, and hydrophobicity constants. No new insight on the role of physiochemical properties of the sulfonamides has resulted. Acid dissociation appears to play a predominant role, since it affects aqueous solubiUty, partition coefficient and transport across membranes, protein binding, tubular secretion, and reabsorption in the kidneys. An exhaustive discussion of these studies has been provided (10). 

The three-dimensional structure of the bacterial membrane protein, bac-teriorhodopsin, was the first to be obtained from electron microscopy of two-dimensional crystals. This method is now being successfully applied to several other membrane-bound proteins. 

The first two volumes in the series New Comprehensive Biochemistry appeared in 1981. Volume 1 dealt with membrane structure and Volume 2 with membrane transport. The editors of the last volume (the present editor being one of them) tried to provide an overview of the state of the art of the research in that field. Most of the chapters dealt with kinetic approaches aiming to understand the mechanism of the various types of transport of ions and metabolites across biological membranes. Although these methods have not lost their significance, the development of molecular biological techniques and their application in this field has given to the area of membrane transport such a new dimension that the appearance of a volume in the series New Comprehensive Biochemistry devoted to molecular aspects of membrane proteins is warranted. 

In the case of PS II membrane proteins, as discussed above, the hydrophobic and hydrophilic pairs of attached lipids can partially support the protein complex at the air-water interface, despite their large size and density. However, in the case of PS II core complex, the detergent strips the attached lipids and some extrinsic proteins. The remaining protein complex is water soluble. It is very difficult to prepare a stable monolayer of water-soluble proteins with the Langmuir method. Indeed, it is hard to directly prepare a stable monolayer of PS II core complex because of its water solubility as well as density. One possible solution is to change the density and ionic strength of the subphase [9]. 

Yano Y, Matsuzaki K (2009) Tag-probe labeling methods for live-cell imaging of membrane proteins. Biochim Biophys Acta-Biomembranes 1788 2124—2131... 

It is important that the method used to detach cells from their growing surface is compatible with end use. For final use as cell control material, it is important to use a methodology that preserves structural integrity and membrane protein localization. Enzymatic-based reagents may affect proteins on the surface of cells. 

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

This is an important observation given that plasma membrane proteins are often used as markers of disease. This experiment demonstrated that shotgun proteomic analysis could be successfully performed on microdis-sected, formalin-fixed tissues using the antigen retrieval method with a sensitivity equal to that of analysis of the soluble fraction of a fresh-frozen sample. 

The last sample preparation method for IMS is the transfer of a tissue section onto the PVDF membrane. Proteins in the section can be transferred onto the PVDF membrane and then analyzed on the membrane. The advantage of this method is that the enzyme can be digested for MS" measurement, because the information on protein localization in the organization is fixed on the membrane.5,20 This technique can denature, reduce, and digest the proteins in the tissue section efficiently and remove the salt from the tissue. This increases the efficiency with which biological molecules are ionized, making it possible to obtain sensitive mass imaging spectra. 

Copeland S., Siddiqui J., Remick D., Direct comparison of traditional ELISAs and membrane protein arrays for detection and quantification of human cytokines, J Immunol Methods 2004 284 99-106. 

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. 

Zeheb, R., Chang, V., and Orr, G.A. (1983) An analytical method for the selective retrieval of iminobi-otin-derivatized plasma membrane proteins. Anal. Biochem. 129, 156-161. 

Henry GD, Sykes BD (1994) Methods to study membrane protein structure in solution. Methods Enzymol 239 515-535... 

The existence of two distinct functions within the one membrane protein raises a number of interesting questions about transporter functions, such as what is the relative importance of the two functions Can they be allosterically regulated under physiological conditions or manipulated by pharmacological methods What is the structural basis for the dual functions In the next section, we shall attempt to address some of these issues and also to present some of the current understanding of the structural basis for these functional states. 

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