The Protein Protocols Handbook
Since the publication of the bestselling second edition of John Walker's widely acclaimed Protein Protocols Handbook, there have been continual methodological developments in the field of protein chemistry. This greatly enhanced third edition i
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1. Introduction Chemical modifications of proteins may be performed simply and rapidly to provide preliminary data regarding the role of particular amino acids in a given protein. Many reviews and books cover these aspects of protein chemistry; only a few are cited here (1–4). In particular, the book by Means and Feeney (1), although about 30 yr old, is an excellent introduction and a practical guide to this field. Notably, even in the era of molecular biology, when site-directed mutagenesis has become widely accessible (including with nonnatural amino acids), selective chemical modifications are still applied regularly. In most cases, chemical modifications are used, often together with site-directed mutagenesis, to either identify or confirm the role of active site residues (for recent examples see ref. 5–9). But chemical modifications are also applied for the generation of improved and modified proteins for a variety of applications (10–12). More recent applications in the field of chemical biology include proteomics (e.g. protein arrays and mass spectroscopy analyses; ref. 13), and a broad range of side-chain modifications, including groups that mimic posttranslational modifications (14–16), and selective chemical modifications of proteins that make use of specific enzymes (17,18). The modifications discussed in the following chapters are side chain selective, that is, under appropriate conditions, the reagents mentioned in this chapter (and additional reagents mentioned in refs. 1–4) react specifically with a single type of amino acid side chain. Hence, loss of activity (enzymatic, binding, or other biological activity) following treatment of the protein with such a
From: The Protein Protocols Handbook, Third Edition Edited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC 2009
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modifying reagent is considered to be an indication for the role of that side chain in the active site of the protein. Data obtained by side chain modifications must be analyzed with caution (as is the case for data obtained by genetic site-directed mutagenesis). Loss of activity on treatment with a reagent might be the result of conformational changes or other changes that occur far from the active site. Some of the reagents, in particular when applied in large excess or under inappropriate conditions, may react with more than one type of side chain or may even disrupt the overall fold of the protein. In general, the type of modifications that alter the size of a particular residue, but not its charge, are preferred (see Chapters 90 and 91). In addition, the reactivity of a certain type of side chain in a protein varies by several orders of magnitude owing to interactions with neighboring groups that affect the accessibility and reactivity. For example, the pKa of the carboxylate side chain of aspartic acid is generally approx 4.5; however, interactions with other side chains may increase the pKa by more than three units. This change will have a major effect on the reactivity of such
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