(221b) Single Molecule Force Measurements: New Insight into Macromolecules
Single molecule force measurement using Atomic Force Microscopy (AFM) is a relatively new tool used to investigate interaction forces between nanometer-scale objects. In this work we have used this technique for analyzing two different biological molecules: inteins with well characterized secondary structure and NSP1 (FG-Nups) that have little or no secondary structure. Inteins are self-splicing and self-cleavage elements that exist as in-frame protein fusions with two flanking protein fragments called exteins. Combining protein engineering with random mutagenesis, small mutant inteins with enhanced splicing or cleavage properties have been isolated. Inteins have been used in affinity fusion-based protein purification as linkers that can be triggered to splice or cleave through excursions in pH or temperature. Results of both AFM imaging of inteins during slicing and single molecule force measurements are presented.