Elastomeric proteins are essential in various biological processes, and many of them exist in parallel dimers or multimers. For example, in striated muscle sarcomeres, the elastic I-band regions of the giant muscle protein titin are arranged into a hexameric structure to mediate the passive elasticity of muscles. However, approaches are limited in studying the mechanical stabilities of these systems. Our recently developed atomic force microscopy (AFM)-based two-molecule force spectroscopy (TMFS) technique has made it possible to investigate the mechanical properties two parallelly arranged elastomeric proteins. Here, we use TMFS to characterize the mechanical unfolding of the parallel dimer of NuG2 polyprotein. NuG2 is the fast-folding variant of the B1 IgG binding domain of protein G. We used SpyTag-SpyCatcher based chemistry to engineer a parallel dimer of (NuG2) 4 , which was picked up and stretched by using AFM. The mechanical unfolding of parallel dimers of NuG2 was characterized by using constant pulling-speed and constant loading-rate experiments. The unique mechanical fingerprints and unfolding kinetics of the parallel dimers of (NuG2) 4 were characterized in detail. Our results showed that the parallelly arranged (NuG2) 4 behave largely independently, and the mechanical unfolding force of the parallel dimer is smaller than 2 times of a single NuG2. Our work paves the way to characterize the mechanical unfolding of parallelly arranged multimers.
Liu et al. (Sun,) studied this question.