Why Are Proteins Charged? Networks of Charge–Charge Interactions in Proteins Measured by Charge Ladders and Capillary Electrophoresis

Abstract

Almost all proteins contain charged amino acids. While the function in catalysis or binding of individual charges in the active site can often be identified, it is less clear how to assign function to charges beyond this region. Are they necessary for solubility? For reasons other than solubility? Can manipulating these charges change the properties of proteins? A combination of capillary electrophoresis (CE) and protein charge ladders makes it possible to study the roles of charged residues on the surface of proteins outside the active site. This method involves chemical modification of those residues to generate a large number of derivatives of the protein that differ in charge. CE separates those derivatives into groups with the same number of modified charged groups. By studying the influence of charge on the properties of proteins using charge ladders, it is possible to estimate the net charge and hydrodynamic radius and to infer the role of charged residues in ligand binding and protein folding.

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

“…Previous studies have attributed the infrequent participation of Lys in interfaces to the entropic cost of restricting its highly mobile side chain. 14,28 This proposal seems to be incompatible with our results (for the specific but very well-defined The results of this study also emphasize the significant role of surface charges in mediating interactions between proteins, 29,30 and suggest that charges, as a constraint in determining proteinprotein interfaces, might be modulated (or reduced) to control biomolecular recognition. We showed that acetylation of positively charged lysine residues gives rise to new protein-protein interfaces, which are less complementary electrostatically but which involve contacts that are more well-defined geometrically.…”

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…The electrophoretic mobility, μ (eq 11), of a molecule is proportional to its charge, Z, and inversely proportional to its molecular weight, M ,395 where the exponent α relates the molecular weight of the protein to its hydrodynamic drag and C P is a proportionality constant. 394,[396][397][398] Equation 11 shows that a change in the electrophoretic mobility of the protein can be altered by a ligand that changes (i) the charge of the receptor, (ii) the hydrodynamic drag of the receptor, or (iii) both. (11) When CA binds to an arylsulfonamide, the change in the hydrodynamic drag of the protein is negligible, and the assay relies on having a charged ligand to produce a significant change in the charge of the protein-ligand aggregate and, thus, a change in the electrophoretic mobility of the receptor ( Figure 9A).…”

“…(12) A Scatchard plot of Δμ P,L /[L] versus Δμ P,L results in a line with slope equal in magnitude to the binding constant, where Δμ P,L is the difference between the mobility of the protein (P) at a given concentration of ligand, [L], and the mobility the protein without the ligand. 394,[396][397][398] Determination of the binding constant of CA to neutral ligands requires a competitive binding assay because small neutral ligands do not cause a shift in the mobility of the protein. The competitive assay involves the addition of a neutral ligand to a sample of CA containing a fixed concentration of a charged ligand of known affinity.…”

“…The investigators attributed this lack of success to the fact that it is more difficult to engineer hydrogen bonds and ionic interactions than hydrophobic contacts; 507 this idea is consistent with the observation that hydrogen bonds and ionic interactions are often geometrically demanding. 4,30,397 Boriack et al also examined the affinity of polar or charged amino acids as secondary recognition elements, but their amino acids were linked to the benzenesulfonamide scaffold by triethylene glycol tails (86)(87)(88). 185 (253) is of historical importance.…”

“…397 In conjunction with CE and affinity capillary electrophoresis (ACE, see section 8.4.2), charge ladders are able to provide quantitative information on these electrostatic effects. Protein charge ladders and ACE were developed using CA as the model protein.…”