Combining Single Molecule Optical Trapping and Small Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein Alpha-Helix

Abstract

A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The stability of the ER/K alpha -helix arises from the charge-charge interactions between its glutamic acid (E) and Arginine (R) or Lysine (K) side chains. The flexibility of the ER/K alpha-helix determines whether it behaves as a force-transducer, rigid spacer or flexible linker in proteins. The ER/K alpha-helix spans long distances with relatively few amino acid residues, has known salt and temperature sensitivity, and can be expressed in E. coli, making it an important tool in engineering proteins, provided its mechanical properties are clearly established. We have quantified the flexibility of the ER/K alpha-helix in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small angle x-ray scattering (SAXS), combined with Montecarlo simulations to demonstrate that the ER/K alpha-helix behaves as a worm-like-chain with persistence length of ∼ 15 nm. This persistence length is dependent on the relative content of R and K residues in the ER/K alpha-helix. Knowledge of the persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force-transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch motif containing protein.