Methods for studying protein

The advantages of resonance Raman spectroscopy have already been discussed in section BL2.2.3. For these reasons it is rapidly becoming the method of choice for studying large molecules in solution. Flere we will present one study that exemplifies its attributes. There are two complementary methods for studying proteins. 

MC Meyer, DE Guttman. Dynamic dialysis as a method for studying protein binding. II Evaluation of the method with a number of binding systems. J Pharm Sci 59 39-48, 1970. 

Severinov, K., and Muir, T.W. (1998) Expressed protein ligation, a novel method for studying protein-protein interactions in transcription./. Biol. Chem. 273(26), 16205-16209. 

B. Somogyi, S. Papp, A. Rosenberg, I. Seres, J. Matko, G. R. Welch, and P. Nagy, A doublequenching method for studying protein dynamics Separation of the fluorescence quenching parameters characteristic of solvent-exposed and solvent-masked fluorophores, Biochemistry 24, 6674-6679 (1985). 

The advent of recombinant DNA technology has led to an increased interest in the structural characterization of proteins by spectroscopic methods. Few spectroscopic techniques can provide the amount of information regarding protein secondary and tertiary structure which can be obtained from circular dichroism (CD) spectroscopy. In this chapter we describe the capabilities of CD spectroscopy to provide details on the globular structure of proteins. In addition, we will provide an overview of quantitative secondary structure estimates via CD spectroscopy and of specialized CD methods for studying proteins in contact with membranes and other biomolecules. Certain aspects of protein CD spectroscopy have been previously reviewed [1-19]. 

When Sanger and colleagues began their work in 1945, it was known that insulin was a small protein consisting of two or four polypeptide chains linked by disulfide bonds. Sanger and his coworkers had developed a few simple methods for studying protein sequences. 

Western blot analysis. As noted earlier for Northern blotting, Western blotting also plays on the name Southern and is a popular molecular biology method for studying proteins using antibodies and corresponding markers. 

Prestrelski et al. [34] demonstrated that FTIR is a rapid and useful method for studying protein conformation in the dried state and can aid in determining the optimum conditions for stabilization of proteins during freeze-drying. 

Fluorescent spectroscopy, because of its high level of sensitivity, has long been a powerful method for studying protein behavior. Site-specific attachment of fluorophores to a unique cysteine in a protein of interest is a traditional route for the production of fluorescent proteins. In addition, the discovery of fluorescent proteins, such as the green fluorescent protein (GFP) from the jellyfish Aequorea victoria [62], has provided a genetic approach for the production of fluorescently labeled proteins. Both these methods, however,... 

TIRF occupies a unique niche, providing a noninvasive method for studying protein adsorption in situ and in real time. The TIRF technique, as it is applied in our laboratory, has been used successfully to study both macroscopic and molecular aspects of protein adsorption. The present goal of this laboratory is to elucidate the interactions occurring when a protein adsorbs to a solid surface using the TIRF technique. It is hoped that, eventually, a complete, general description of the protein adsorption process will be attained. 

Huang, N. and Jacobson, M.P. (2007) Physics-based methods for studying protein-ligand interactions. Current Opinion in Drug Discovery e[ Development, 10, 325-331. 

Duim, G. A. Dobbie, I. M. Monypeimy, J. Holt, M. R. Zicha, D. Fluorescence localization after photobleaching (FLAP) a new method for studying protein dynamics in living cells. J. Microsc. 2002, 205, 109-112. 

Marintchev, A., Frueh, D., and Wagner, G. 2007. NMR methods for studying protein-protein interactions involved in translation initiation. Methods Enzymol. 430 283-331. 

Nondenaturing Polyacrylamide Gel Electrophoresis (NPAGE) as a Method for Studying Protein Interactions... 

To summarize, the advantages of fluorescence methods for studying protein unfolding reactions are the wide concentration range that can be measured and the sensitivity of the signal to the microenvironment of the fluorophore. Also, fluorescence signals of the native and unfolded state can provide some low resolution structural information about these states (at least with respect to the microenvironment of the fluorophores). 

Hlady, V., Buijs, J., Jennissen, H.P. Methods for studying protein adsorption. Methods Enz5rmology 309,402-429 (1999)... 

Circular dichroism A very commonly used method for studying protein conformational changes. 

Fluorescence The most sensitive of the commonly used optical methods for studying protein unfolding transitions. 

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