Distinct solid and solution state self-assembly pathways of RADA16-I designer peptide

Abstract

Solid state NMR measurements on selectively (13) C-labeled RADA16-I peptide (COCH3 -RADARADARADARADA-NH2 ) were used to obtain new molecular level information on the conversion of α-helices to β-sheets through self-assembly in the solid state with increasing temperature. Isotopic labeling at the A4 Cβ site enabled rapid detection of (13) C NMR signals. Heating to 344-363 K with simultaneous NMR detection allowed production of samples with systematic variation of α-helix and β-strand content. These samples were then probed at room temperature for intermolecular (13) C-(13) C nuclear dipolar couplings with the PITHIRDS-CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X-ray diffraction. Independence of PITHIRDS-CT decay shapes on overall α-helical and β-strand content infers that β-strands are not observed without association with β-sheets, indicating that β-sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS-CT NMR data were compared with results of similar measurements on RADA16-I nanofibers produced by self-assembly in aqueous salt solution. We report that β-sheets formed through self-assembly in the solid state have a structure that differs from those formed through self-assembly in the solution state. Specifically, solid state RADA16-I self-assembly produces in-register parallel β-sheets, whereas nanofibers are composed of stacked parallel β-sheets with registry shifts between adjacent β-strands in each β-sheet. These results provide evidence for environment-dependent self-assembly mechanisms for RADA16-I β-sheets as well as new constraints on solid state self-assembled structures, which must be avoided to maximize solution solubility and nanofiber yields.

Keywords: designer peptide; isotopic labeling; self-assembling peptide; solid state NMR; structural studies.