Using Covalent Dimers of Human Carbonic Anhydrase II To Model Bivalency in Immunoglobulins

Abstract

This paper describes the development of a new bivalent system comprising synthetic dimers of carbonic anhydrase linked chemically through thiol groups of cysteine residues introduced by site-directed mutagenesis. These compounds serve as models with which to study the interaction of bivalent proteins with ligands presented at the surface of mixed self-assembled monolayers (SAMs). Monovalent carbonic anhydrase (CA) binds to benzenesulfonamide ligands presented on the surface of the SAM with Kdsurf = 89 nM. The synthetic bivalent proteins—inspired by the structure of immunoglobulins—bind bivalently to the sulfonamide-functionalized SAMs with low nanomolar avidities (Kdavidity, surf = 1 – 3 nM); this difference represents a ~50-fold enhancement of bivalent over monovalent association. The paper describes dimers of CA having i) different lengths of the covalent linker that joined the two proteins, and ii) different points of attachment of the linker to the protein (either near the active site (C133) or distal to the active site (C185)). Comparison of the thermodynamics of their interactions with SAMs presenting arylsulfonamide groups demonstrated that varying the length of the linker between the molecules of CA had virtually no effect on the rate or avidity of association, or on the avidity of these dimers with ligand-presenting surfaces. Varying the point of attachment of the linker between monomeric CA’s also had almost no effect on the avidity of the dimers, although changing the point of attachment affected the rates of binding and unbinding. These observations indicate that the avidity of these bivalent proteins, and by inference the avidities of structurally similar bivalent proteins such as IgG, are unexpectedly insensitive to the structure of the linker connecting them.

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacteriumThiomicrospira crunogenaXCL-2: insights into engineering thermostable enzymes for CO2sequestration

“…4d). Previous studies have shown that hCA II can be engineered into a dimer via an intermolecular disulfide (Mack et al, 2011); however, this study did not speculate on the potential increased stability of the hCA II dimer. The presence of an engineered disulfide will potentially keep the hCA II dimer intact at more extreme pH levels (pH of $3.0; He et al, 2006).…”

Dependence of Avidity on Linker Length for a Bivalent Ligand–Bivalent Receptor Model System

“…3 This assay consists of four steps (Figure 3a). i) We allowed a solution of (CA) 2 and LRL to equilibrate in a stirred quartz cuvette while the temperature was maintained at 25 ± 1 °C, and observed the fluorescence of the tryptophan residues of the protein (λ ex = 280 nm, λ em = 340 nm).…”

“…3 This theory predicts the change in free energy (Δ G ° avidity ) for the bivalent association of (CA) 2 to bivalent ligands would be twice the change in enthalpy for the monovalent interaction (Δ H ° mono ) plus the change in entropy for a single monovalent interaction (TΔ S ° mono ) (eq 1). 8 In this work, we designed and synthesized a series of bivalent ligands (LRL) with…”

“…We can easily change the nature of the linkers (length and flexibility) connecting the binding sites in the dimers. 3 These sorts of changes would require major efforts in mutagenesis to carry out on any IgG. ii) The carbonic anhydrases (HCA I (28.8 kDa), HCA II (29.2 kDa), and bovine carbonic anhydrase BCA II (29.1 kDa), which differ only by 382 Da) are similar in size to the Fabs of IgG (~50 kDa).…”

“…3 We prepared three dimers of CA having different lengths of the oligo(ethylene glycol) linker that joined the two molecules of CA, and two dimers with different points of attachment of the linker to the protein. We found that the dimers of CA bound bivalently to the SAM with low nanomolar avidities ( K d avidity, surf = 1 − 3 nM, M eff = 3 to 20 μM) while monomeric carbonic anhydrase (CA) bound to the ligands of the SAM less strongly ( K d surf = 89 nM).…”