title: Complete noise analysis of a simple force spectroscopy AFM setup and its applications to study nanomechanics of mammalian Notch 1 protein.
abstract: We describe a complete noise analysis and application of a custom made AFM force spectroscopy setup on pulling a recombinant protein with an NRR domain of mouse Notch 1. Our table top AFM setup is affordable, has an open architecture, and is easily transferable to other laboratories. Its calculated noise characteristics are dominated by the Brownian noise with 2% non-Brownian components integrated over the first thermally induced resonance of a typical cantilever. For a typical SiN cantilever with a force constant of ~15 pN nm(-1) and in water the force sensitivity and resolution are less than 10 pN, and the corresponding deflection sensitivities are less than 100 pm Hz(-1/2). Also, we obtain a sub-ms time resolution in detecting the protein length change, and only few ms cantilever response times as measured in the force clamp mode on a well-known protein standard. Using this setup we investigate force-induced conformational transitions in the NRR region of a mouse Notch 1. Notch is an important protein related to leukemia and breast cancers in humans. We demonstrate that it is feasible to develop AFM-based studies of the force-induced conformational transitions in Notch. Our results match recent steered molecular dynamics simulations of the NRR unfolding and constitute a first step towards a detailed study of Notch activation with AFM.
authors.affiliation: Department of Physics, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Ashim
authors.initials: A
authors.lastname: Dey
authors.forename: Robert
authors.initials: R
authors.lastname: Szoszkiewicz
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena / Q000502:physiology; D051379:Mice; D018625:Microscopy, Atomic Force / Q000295:instrumentation* / Q000379:methods*; D056004:Molecular Dynamics Simulation; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D011487:Protein Conformation; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; D011994:Recombinant Proteins