AbstractAtomic force microscopy (AFM) is an important tool for high resolution studies in biophysics and mechanical studies directed at biological materials. A strong suit of AFM is its ability to measure hardness/elasticity, nonspecific adhesion or ligand-receptor interactions at the picoNewton scale. Molecular interactions are critical factors in a variety of biological phenomenon; such as the initiation, modulation and termination of DNA replication, transcription, enzyme activity, infection, immune responses, tissue generation, wound healing, cell differentiation, apotopsis and physiological responses from drugs, hormones or toxic agents. Using AFM, scientists can probe and quantify these interactions in their native, liquid environments at physiological pH or perform dynamic experiments in situ by removing or adding ions, solutes and reagents to the sample environment. Bioconjugation chemistry and surface chemistry are crucial because a selective ligand must be immobilized on the tip of an AFM probe so that the AFM can resolve the mechanical force required to separate the ligand and its target. The resulting data can be used to calculate forces of unbinding, derive rate constants and infer structural information about the binding pocket. Biomolecular recognition experiments with AFM can be greatly enhanced through the use of relatively short (~8-10 nm), heterobifunctional, elastic, polyethylene glycol (PEG) linkers to immobilize ligands. Heterobifunctional linkers are used in order to permit their sequential immobilization and bioconjugation, while minimizing undesirable polymerizations or self-conjugation. The linkers have an N-hydroxysuccinimide ester at one end to permit their attachment to aminated silicon or silicon nitride AFM probes. Other reactive functional groups, such as a biotin, maleimide, disulfide, aldehyde, or a photoreactive group reside at the opposite end of the linker to permit the direct or indirect attachment of intact antibodies, Fab fragments, peptides, nucleic acids or other biological entities. The PEG linkers are flexible, so an attached ligand has freedom to diffuse within a defined volume of space and approach the binding site in a thermodynamically favorable manner. PicoTREC, an accessory for the Agilent AFM, uses ligand-PEG modified cantilevers to generate a topography image and a recognition image of biomolecular interactions. As the modified cantilever gently oscillates at defined amplitude, it is scanned across a sample and PicoTREC converts the information derived from ligand-receptor interactions into a high resolution, nanometer-scale map. Consequently, the locations of discrete molecular interactions can be easily determined and compared with a topography image of the sample.
Structural Insights into Selective Ligand–Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists