A Versatile Polypeptide Platform for Integrated Recognition and Reporting: Affinity Arrays for Protein–Ligand Interaction Analysis

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Abstract
A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target‐protein binding. The platform is based on a versatile 42‐residue helix–loop–helix polypeptide that dimerizes to form four‐helix bundles and allows site‐selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well‐characterized interaction between the model target‐protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof‐of‐concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02–3 μM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target‐protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform‐expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein‐microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.