Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

Chemical protein modification
Features of the chemical modification method

Chemical modification, i.e., carrying out Chemical Reactions that alter the Covalent Structure of a protein, is one of the most important techniques used to characterize protein functional groups. For a long time, this method dominated The Study of structure-function relationships in Proteins; however, it has recently lost its monopoly, yielding in many respects to the rapidly developing Site-Directed Mutagenesis. Nevertheless, it remains fully relevant as a primary screening tool for functionally significant groups within a protein molecule, particularly those endowed with anomalous reactivity (although site-directed mutagenesis holds significant advantages at the final stages of research).

At the same time, chemical modification reactions form the basis for creating irreversible Enzyme Inhibitors, are used for the immobilization of proteins (especially Enzymes and Antibodies) in biotechnological Applications, and serve to study the topography of protein surfaces and their localization within complex structures, such as membranes and other supramolecular assemblies.

Protein chemical modification reactions are extremely numerous. Only a few of the most representative ones will be discussed below.

When discussing the FEATURES OF THE method, one must first emphasize the striking Specificity of proteins as objects of chemical modification. Often, upon embarking on chemical modification, a researcher is forced to proceed from a simplified view of a protein as a carrier of a specific set of functional groups belonging to the side chains of amino acid residues. In this approach, The chemical properties of the latter are essentially equated with the well-described and fairly simply predictable properties of corresponding groups in low-molecular-weight compounds, particularly in Peptides and Amino Acids. The Overview of Reagents used for protein modification given below is essentially based on this very simplification.

However, one should not overlook the limitations of this approach. Indeed, some of the functional groups, mainly those located On the surface of the globule, are surrounded by Water and do not participate in contact networks with other protein groups. To a reasonable approximation, they can be considered analogous in reactivity to the same groups in small molecules, such as peptides. However, the number of such "canonical" groups is not all that large and, more importantly, they are not what determines the chemical specificity and unique Nature of the protein molecule.

Three main reasons can be cited for the radical alteration of the Chemical Activity of functional groups incorporated into a protein macromolecule.

1. Some functional groups are concealed within the interior of the globule and are therefore inaccessible to chemical reagents (as long as the protein maintains its native structure). For example, in a relatively small protein—Carboxypeptidase A—one Tyrosine and two Tryptophan residues are completely buried inside the globule; 13 tyrosine and 6 tryptophan residues reside in the surface layer and are partially accessible to the solvent. Only four tyrosine residues are immersed in the water surrounding the protein and can exhibit the same chemical properties as tyrosine in small peptides. Chemical modification reactions designed to achieve complete substitution of all accessible groups of a given type are used to count and identify buried functional groups.

2. Many functional groups contact neighboring groups within the protein, forming distinctive ensembles. Participation in such ensembles can strongly, sometimes beyond recognition, alter the reactivity of the partners. This is confirmed, in particular, by the fact that dissociation constants and their corresponding pK_a values for ionogenic protein groups span very wide ranges; in other words, the acid-base properties of these groups heavily depend on their microenvironment.

Thus, in small peptides, the ω-carboxyl groups of the side chains of aspartic and glutamic acids have a pK_a close to 4.7, being similar in this respect to the carboxyl groups of acetic and other aliphatic carboxylic acids. Across the entire population of proteins, however, their pK_a values span a huge range: from 1.5 (one of the carboxyl groups in the active center of Pepsin) to 7.5 (the carboxyl group of the Glu-49 residue in the α-subunit of tryptophan synthase). Thus, in proteins, carboxyl groups can act both as very strong acid groups—almost completely dissociated in the physiological pH range—and as very weak ones, barely half-dissociated even in a neutral medium, with their dissociation constants varying by six orders of magnitude.

The change in the reactivity of a given functional group can be caused not only by its interaction with another group (e.g., with a second carboxyl group, where one becomes a strong acid and the other a weak one), but also by a local change in the polarity of the group's microenvironment. For example, it is believed that the immersion of the β-carboxyl group of the Glu-35 residue into a hydrophobic region of hen egg-white Lysozyme hinders proton dissociation because the resulting anion cannot be stabilized by Hydration; the pK_a of this group is close to 6.

3. The surface of the protein globule is far from indifferent to the reagents used in modification. Even if these reagents do not contain specially tailored structures that promote selective binding to a specific zone of the protein (so-called affinity modification), they are very likely to be bound by the protein through interactions with charged groups, Hydrophobic surface patches, Hydrogen bond Donors or acceptors, etc. Thus, the actual modification is preceded by the binding of the reagent to a specific region of the Cell/13.html">Protein Structure. This effectively leads to the local concentration of the reagent at that point on the protein molecule and sharply increases the probability of reaction for functional groups that may happen to be adjacent to the reagent-binding site.

It is easy to see that the aforementioned Features of protein behavior in chemical modification reactions are not accidental, but reflect the Fundamental properties of its structural Organization and function. From a methodological standpoint, this means that when interpreting the results of chemical modification, one cannot rely on data regarding the reactivity of corresponding groups in model compounds. Strictly speaking, in each case—after carrying out the reaction and isolating the modification product (or separating them, if there are several)—direct Methods must be used to establish precisely which Functional groups of the protein were modified and to determine which amino acid residues in the polypeptide chain they belong to. Thus, the subsequent overview of reagents and their reactions with protein functional groups provides only a general orientation regarding the potential Applications of the chemical modification method.