Enzyme-mediated polyprotein conjugation for single-molecule force spectroscopy

Single-molecule force spectroscopy (SMFS) is a powerful tool for investigating the energy landscapes of protein folding and unfolding. However, performing precise measurements with SMFS-based tools such as an atomic force microscope (AFM) is challenging due to nonspecific interactions between the AFM tip and sample, which obscure signatures of proteins of interest in force–extension curves. Polyproteins comprising multiple, covalently linked proteins often improve the statistics of single-molecule AFM measurements and provide fingerprints to filter force–extension curves from nonspecific interactions. We demonstrate enzyme-mediated polyprotein synthesis of ion-responsive proteins in a one-pot reaction using Sortase A. We monitor polyprotein reactions using polyacrylamide gel electrophoresis and validate polyprotein products using ion-exchange chromatography and MALDI mass spectroscopy. Ion-responsive polyproteins with varying amino acid sequences enable investigations with single-molecule AFM to probe the sequence dependence of ion-driven protein folding.