(656f) Single Molecule Characterization of Designed Ankyrin-Repeat Protein/Ligand Interaction

A fundamental understanding of biomolecular recognition is essential to the determination and optimization of important design parameters for developing the next generation of biosensors and biothreat detection systems. Antibodies are the most widely used binding molecules in biomedical research with high specificity and affinity to the targets of interest; however, they are generally expensive to produce and require disulfides for stability. To overcome these limitations, non-antibody scaffolds such as designed ankyrin repeat proteins (DARPins) have been developed as binding proteins. DARPins have been engineered to detect diverse target proteins with pM dissociation constant, thus offering an attractive alternative to antibodies. Combining protein engineering techniques with single-molecule force spectroscopy, we have developed techniques for characterizing the binding kinetics of the DARPin off7 and maltose binding protein (MBP) bond under external forces. MBP was directionally oriented onto a lipid bilayer platform by attaching the CD44 transmembrane domain to its N-terminus. Atomic force microscopy (AFM) was performed to measure the rupture force and dissociation kinetic constants of the interaction between off7 and MBP. Rupture force (f_b) of a single off7-MBP bond was determined to be a function of loading rate and was in the range of 50-80 pN. The unstressed off-rate (k⁰_off) and reactive compliance (x_β) of off7-MBP bond were calculated to be 6.1x10^-3 /s and 0.59 nm, respectively. By analyzing the crystal structure of the off7-MBP complex using virtual alanine scanning in the Robetta server, binding energy at the interfacial amino acids of the DARPin off7 protein was estimated. Based on this simulation result, we selectively mutated interfacial amino acids and measured their effects on the binding kinetics. Our data show that mutations at two specific amino acids on DARPin off7 essentially abrogate binding. Therefore, the DARPin off7-MBP binding interface is characterized by a hot-spot motif in which binding energy is largely concentrated only to a few amino acids. In summary, we have developed a novel experimental approach to study the kinetics (k⁰_off) and micromechanical properties (f_b and x_β) of DARPin off7-MBP bond under forced dissociation. Our data provide informative molecular cues to understand how the engineered protein interacts with the target molecule.