Localizing Chemical Groups while Imaging Single Native Proteins by High-Resolution Atomic Force Microscopy

Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nano-technological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force-distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni2+-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni2+-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites.