The Study of Proteins

Many complex systems have been spread on liquid interfaces for a variety of reasons. We begin this chapter with a discussion of the behavior of synthetic polymers at the liquid-air interface. Most of these systems are linear macromolecules however, rigid-rod polymers and more complex structures are of interest for potential optoelectronic applications. Biological macromolecules are spread at the liquid-vapor interface to fabricate sensors and other biomedical devices. In addition, the study of proteins at the air-water interface yields important information on enzymatic recognition, and membrane protein behavior. We touch on other biological systems, namely, phospholipids and cholesterol monolayers. These systems are so widely and routinely studied these days that they were also mentioned in some detail in Chapter IV. The closely related matter of bilayers and vesicles is also briefly addressed. 

Flolmes K C and Blow D M 1965 The use of x-ray diffraotion in the study of protein and nuoleio aoid struoture Meth. Biochem. Anal. 13 113-239... 

Fisher T E, Oberhauser A F, Carrion-Vazquez M, Marszalek P E and Fernandez J M 1999 The study of protein mechanics with the atomic force microscope Trends Biochem. Sci. 24 379-84... 

Such globed master equations have recently gained attention in the study of protein folding. See for example C. De Dominicis, H. Orland and F. Lainee, J. Phys. Lett., 46 L463, 1985 E.I. Shakhnovich and A. M. Gutin, Eurphys. Lett., 9 569, 1989 J. G. Saven, J. Wang and P. G. Wolynes, J. Chem. Phys., 101 11037, 1994. 

With the increase in hardware and software, larger systems can be handled with higher accuracy. Much work will continue to be devoted to the study of proteins and polynucleotides (DNA and RNA), and particularly their interactions with more sophisticated methods. Remember proteins and genes are chemical compounds and sophisticated theoretical and chemoinformatics methods should be applied to their study - in addition to the methods developed by bioinfor-maticians. 

The tissue to be analyzed is placed directiy onto the gel. Using the tissue itself and not tissue extracts has advanced the study of proteins that are difficult to extract from tissue, or are damaged by the extraction procedure. Dtif is an important advancement in the area of sample handling and appHcation where direct appHcation of a soHd to a gel matrix may actually enhance resolution. 

An aerial view of the European Synchrotron Radiation Facility at Grenoble, France, an advanced source of synchrotron x-ray radiation for use in the study of protein structure, as well as for use in the physical and material sciences. The synchrotron radiation is produced in the circular building in the lower left of the photograph. (Courtesy of ESRF.)... 

The use of MS-MS to provide sequence information has been described [13] for the study of proteins extracted from yeast (Saccharomyces cerevisiae). The procedure was somewhat complex and consisted of the following steps ... 

TO THE STUDY OF PROTEIN CONFORMATION AT THE OIL-WATER INTERFACE OF EMULSIONS... 

Plaxco, K. W., and Dobson, C. M. (1996). Time-resolved biophysical methods in the study of protein folding. Curr. Opin. Strud. Biol. 6, 630-636. 

The three main applications of VFPs in FRET studies are (1) the study of protein-protein interactions in living cells, (2) the study of conformational changes within a protein, and the (3) use of... 

Sutherland BW, Toews J, Kast J. Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. J. Mass. Spectrom. 2008 43 699-715. 

Thiourea Linkages. Attachment of saccharide units to the surface of PAMAM through thiourea linkages offers one of the most efficient ways to develop multivalent ligands quickly and efficiently for the study of protein-carbohydrate interactions. 

Brockman JM, Fmtos AG, Com RM (1999) A multistep chemical modification procedure to create DNA arrays on gold surfaces for the study of protein-DNA interactions with surface plasmon resonance imaging. J Am Chem Soc 121 8044-8051... 

Dordick, 1991). In addition, Fancy et al. (1996) as well as Fancy and Kodadek (1997, 1998) have applied the oxidation of tyrosine to form dityrosine to the study of protein-protein interactions using nickel-chelated 6 X His tagged fusion proteins in oxidative environments. 

The basic reactions of thiolsulfonates have been known for sometime (Field et al., 1961, 1964), but more recently, they have been applied to the study of protein interactions by site-directed modification of native cysteines or through modification of cysteines introduced at particular points in proteins by mutagenesis. Such studies have yielded insights into the structure and binding site characteristics of proteins (Kirley, 1989). Pascual et al. (1998) used AEAETS to probe the acetylcholine receptor from the extracellular side of the membrane in order to investigate the molecular accessibility and electrostatic potential within the open and closed channel. 

EGS and sulfo-EGS also have been used to study the surface loop motion in FepA (Scott et al., 2002), to characterize the high affinity copper transporter in Saccharomyces cerevi-siae (Pena et al., 2000), and in the study of protein interactions and large protein complexes (Petrotchenko et al., 2005(2005 a or b ). 

Additional information on the use of sulfo-SBED for the study of protein interactions can be found in Chapter 28, Section 3.1. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

Although MTS-ATF-biotin and MTS-ATF-LC-biotin are available commercially (Thermo Fisher and Toronto Research), they are relatively new and don t have the publications or applications backing up their use as sulfo-SBED. A protocol for the use of these compounds in the study of protein interactions can be found in Chapter 28, Section 3.2. 

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... 

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