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Document <strong>Structure</strong>/function studies can take a variety of forms and use a number of techniques including the use of molecular biology, synthetic peptides, and antibodies, or combinations of these methods. Molecular biology is a powerful tool for structure/function analysis. Mutagenesis of cDNAs to produce mutant proteins with point mutations, truncations, or deletions can identify functional sites. One drawback to this approach, especially with large proteins, is the proverbial “needle in a haystack” problem. One has difficulty determining where to begin placing mutations. The synthetic peptide approach can be equally as powerful. One can synthesize individual domains or segments of proteins and test them for agonist or antagonist activities there<strong>by</strong> identifying functional domains. Synthetic peptides can be used to map the epitope specificity of antibodies that block the activity of a protein. Peptides can also be used to produce monospecific antisera to a defined region of a protein. The antibody approach has also proved quite useful in determining functional sites of http://legacy.netlibrary.com/nlreader/nlReader.dll?bookid=12640&filename=Page_437.html (2 of 2) [4/9/2004 12:10:37 AM]
Document Page 438 proteins. One potential disadvantage to using antibodies is the possibility of over interpreting the blocking results because of steric hindrance. A large antibody molecule may inhibit function through binding to a distant site and covering up a functional site. These approaches have all been used with success on a variety of proteins, but are best used in combination. For example, the information obtained from synthetic peptides and antibodies can significantly narrow down the region for site-directed mutagenesis studies. The entire sequence is narrowed to a segment, which reduces the size of the “haystack” in which the needle is hidden. Even though these approaches are powerful methods for determining functional sites on proteins, they are limited if not coupled with some form of structural determination. As Figure 2 illustrates, molecular biology and synthetic peptide/antibody approaches are not only interdependent, they are tied in with structural determination. Structural determination methods can take many forms, from the classic x-ray crystallography and NMR for three-dimensional determination, to two-dimensional methods such as circular dichroism and Fourier Transformed Infrared Spectroscopy, to predictive methods and modeling. A structural analysis is crucial to the interpretation of experimental results obtained from mutational and synthetic peptide/antibody techniques. Figure 2 Flow diagram of structure/function studies. http://legacy.netlibrary.com/nlreader/nlReader.dll?bookid=12640&filename=Page_438.html [4/9/2004 12:10:42 AM]
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