Novel Generation of Crosslinkers allows Single Molecule Force Spectroscopy on Oligomeric Receptors

Abstract

Single molecule force spectroscopy allows for measurement of the unbinding forces between a tip-tethered ligand and a cognate receptor molecule which is immobilized on the sample surface. Conventionally, the atomic force microscope (AFM) tip is aminofunctionalized and the ligand is coupled via a linear heterobifunctional poly(ethylene glycol) (PEG) linker. This approach restricts the use of force microscopy to the investigation of single ligand-receptor pairs. For extension of the technique to the analysis of dimeric or oligomeric receptors, new crosslinkers carrying two or four terminal coupling groups for ligand-attachment were synthesized.The syntheses are based on tri- and pentavalent core units. Heterobivalent PEGs serve as elongations to create the optimal ligand-to-ligand distance for each ligand-receptor system. In case of the bivalent linker, lysine was chosen as branching element and the biotin-streptavidin couple served as test system. The probability density function (histogram) of the unbinding forces showed two peaks, reflecting mono- and bivalent binding of the tip-bound bis-biotin linker to one support-bound streptavidin molecule. The tetravalent linker was prepared from three copies of a symmetric trifunctional subunit formed from lysine which carries a β-alanine on its α-amino group. In a multistep reaction, a 2∗2 “fork” with four terminal amino groups was synthesized. Fortunately it was possible to elongate each prong of the 2∗2 fork with a PEG chain of the same length, using HATU and HOAt for in-situ coupling of amine and carboxyl groups. Terminal azido-groups provide for coupling of alkyne-functionalized ligands directly on the tip. In conclusion, branched crosslinkers permit single molecule force spectroscopy on oligomeric receptors; their modular structure can easily be adapted for different ligand-receptor-system in terms of coupling functions and ligand-to-ligand distance.