Atomic force microscopy captures length phenotypes in single proteins

We use single-protein atomic force microscopy techniques to detect length p henotypes in an Ig module. To gain amino acid resolution, we amplify the me chanical features of a single module by engineering polyproteins composed o f up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded cor e of the module extend 20 Angstrom per module farther than the wild-type po lyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discrimina te the location of the insert and measure its size with a resolution simila r to that of NMR and x-ray crystallography.