Chapter 15 Chemical Modification of Proteins.pdfVIP

CHAPTER 15 Chemical Modification of Proteins INTRODUCTION hemical modifications of reactive amino acid side chains on proteins have many Capplications in modern protein science. Common applications include: fluorescent or radioactive tagging of macromolecules; identifying the subcellular localization of a specific protein, i.e., cell surface, bilayer imbedded, or internal; coupling the protein to another component including another protein, nucleic acids, or a solid support; and as a aid in structural analyses Chapter 11 such as amino acid analysis, sequencing, peptide mapping and mass spectrometry. Another historical application included the use of chemical reagents for structure/function applications, such as identification of residues in enzyme active sites. Over the past decade many, but not all, of these latter approaches have been replaced by site-directed mutagenesis analyses. However, despite the impres- sive power of mutagenesis methods, it is often most productive to utilize both methods in concert—i.e., initial chemical modification studies can be used to identify likely residues involved in a particular biological function, and mutagenesis studies are then used to confirm and extend these initial observations. The most common chemical modifications target the most reactive amino acid side chains and are primarily oxidations, reductions, and nucleophilic or electrophilic substitutions. Chemical reagents that react with side chain amines or carboxyl groups will also react with the terminal amino and carboxyl groups of a protein, respectively, unless these groups were already modified in vivo. Terminal amino and carboxyl groups of a protein are, on average, more reactive than side-chain amino and carboxyl groups, and this difference can in theory be exploited to specifically label these terminal functional groups. Such specific, single-site labeling or attachment would be very useful for a range of applica- t