Protein Protocols Protein Protocols

Chemical Modifications of Proteins 465
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Side Chain Selective Chemical Modifications of Proteins
Dan S. Tawfik
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 non-natural 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).
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 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 67 and 68). 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 a carboxylate group toward the modifying
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
465