Significance of Charge Regulation in the Analysis of Protein Charge Ladders

Abstract

Analysis of protein charge ladders using capillary electrophoresis (CE) provides a method of determining charges of proteins. This method has disregarded the effect of charge compensationsa response of the protein and its environment to a change in electrostatic potential on the surface of the protein. This work examines the difference in charge, ∆Z, between the first two rungs of the ladder of bovine carbonic anhydrase II (BCAII) as a function of pH and ionic strength using CE. These data were analyzed in three ways: using models based on Hückel theory and on charge regulation, and using linear regression. These analyses were in only qualitative agreement, and the differences between them suggest that simple theoretical models for the behavior of colloidal particles cannot establish the value of ∆Z accurately in proteins. Linear regression of mobilities of the rungs of charge ladderssa method proposed in earlier workscontinues to be a computationally convenient method of estimating the charge Z 0 of native proteins, but the accuracy of this method depends on the value of ∆Z. The absolute value of ∆Z cannot presently be established accurately. In the case of BCAII, we suggest ∆Z ) -0.93 for the difference in charge between the first two rungs of the charge ladder at pH ) 8.4 and 10 mM ionic strength. An estimate of the uncertainty in this value for BCAII due to uncertainties in the values of pK a of amino acids and of the hydrodynamic radius is (0.02. Other uncertainties not considered in this analysis will make this value larger. IntroductionA protein charge ladder is a collection of derivatives of a protein generated by converting its charged groups (most commonly lysine -NH 3 + but also aspartate or glutamate -CO 2 -) into electrically neutral ones ( -NHCOCH 3 or -CO 2 CH 3 ). 1 In free solution capillary electrophoresis (CE), these modified proteins separate into distinct peaks or "rungs"; each rung contains regioisomers with approximately the same charge: that is, at least nominally, the same number of modified groups. Charge ladders provide a self-calibrating tool for estimating certain basic physical parameters of proteins such as charge, 2,3 hydrodynamic radius, 4 and electrostatic contributions to the free energy of binding of ligands 5,6 and of protein folding. 7 The simplest analysis of charge ladders assumes that the charge difference (∆Z) between the consecutive rungs of a charge ladder generated by acetylation of lysine -NH 3 + groups is a full unit of charge (∆Z ) -1). Menon and Zydney have made the point that ∆Z may have a value different from -1 if the change in charge of the -amino group on acetylation (which is, in fact, -1, provided that this group is completely protonated before acetylation) is offset by a compensating change in charge elsewhere in the protein. 8 This conceptscharge compensations is well-developed and extensively modeled in colloid chemistry. 9,10 Menon and Zydney suggested that it also applies to proteins and proposed a model to analyze the adjustment of charge of the macro...

Electrophoresis of Protein Charge Ladders: A Comparison of Experiment with Various Continuum Primitive Models

“…The Lee model, described in section II.C, predicts a value of ∆Z EM 47 The accuracy in values of ∆Z EM reflects the accuracy with which electrostatic models predict values of pK a . The accuracy in values of ∆Z BE reflects the accuracy of the experiments and of the mobilities predicted by the BE model.…”

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

“…The increased participation of charged residues in the crystal contacts of [CA-(NHCOCH 3 Aly-perhaps as a result of its charge neutrality, increased surface area, and/or ambidexterity in hydrogen bonding-can more easily accommodate a range of binding partners in crystal contacts.…”

“…We used acetic anhydride to acetylate the lysine residues of CA (this procedure is quantitative; see Supporting Information); we refer to crystals of the peracetylated variant as [CA-(NHCOCH 3 ) 18 ] -19 . Capillary electrophoresis confirmed that all 18 residues were acetylated (Figure S1) 16 ; X-ray crystallography allowed us to determine its structure, and, thus, permitted a comparison with the structure of wild-type CA, 6 which we, hereafter, refer to as [CA-(NH 3 + ) 18 ] -3.…”

“…This comparison allowed us to study protein-protein interactions in which the structures of the contacting proteins is very similar, but in which the number of surface-exposed NH 3 + groups differs by 18, and in which the net charge changes from close to zero (Z = -3.4) to highly negatively charged (Z = -19) (The small discrepancy between the number of lysine groups acetylated and the change in net charge in solution indicates that the removal of positive charges by peracetylation is partially compensated, possibly by changes in the pKa of carboxyl groups on the protein surface). 3,4 The increase in net charge that results from acetylation-an increase that occurs through a decrease in the total number of positive charges-is accompanied by a net decrease (by 18 NH 3 + groups) in the net surface density of charged groups, and also by a change in the surface of the protein from more "polyzwitterionic" to more "polyanionic" upon acetylation.…”

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“…Thus eliminating the positive charge from ε-NH+ group and replacing with negative charge would increase the net negative charge by 2 units. However this change would be less than 2 units due to charge regulation, due to the adjustment of protonation states of other ionizable residues predominantly histidine in a manner that reduces the total change in charge [18,21].…”