Revealing Fast Structural Dynamics in pH-Responsive Peptides with Time-Resolved X-ray Scattering

Abstract

Many biomaterials can adapt to changes in the local biological environment (such as pH, temperature, or ionic composition) in order to regulate function or deliver a payload. Such adaptation to environmental perturbation is typically a hierarchical process that begins with a response at a local structural level and then propagates to supramolecular and macromolecular scales. Understanding fast structural dynamics that occur upon perturbation is important for rational design of functional biomaterials. However, few nanosecond time-resolved methods can probe both intra- and intermolecular scales simultaneously with a high structural resolution. Here, we utilize time-resolved X-ray scattering to probe nanosecond to microsecond structural dynamics of poly-l-glutamic acid undergoing protonation via a pH jump initiated by photoexcitation of a photoacid. Our results provide insights into the protonation-induced hierarchical changes in packing of peptide chains, formation of a helical structure, and the associated collapse of the peptide chain.

Figure 1

Static X-ray solution scattering results for PGA. (a) Log−log plot of X-ray solution scattering data for 200 unit PGA at different pH values. The inset shows the plots on a linear scale. (b) SAXS data for peptide lengths of 20, 50, 100, and 200 residues at pH 7. The inset presents the characteristic distances between chains because of intermolecular repulsion at 2 mg/mL concentration as a function of peptide length calculated from the SAXS peak location.

Figure 2

TRXSS results from photoinduced pH jump experiment on 200 residue PGA. (a) Time series of the scattering difference signals at different time delays. (b) Integrated TRXSS signal as a function of time delay. (c) Species-associated population dynamics of the intermediate and final species following protonation. (d) Species-associated scattering differences derived from global analysis. (e) Comparison of final species-associated difference F_p signal with difference signal derived from static scattering curves at pH = 5.9 and pH = 5.4.

Figure 3

Comparison of integrated WAXS integrated TRXSS signal kinetics for different peptide lengths—200 units (black) and 20 units (green). (Inset) Comparison of extracted lifetimes from kinetic analysis of WAXS-integrated TRXSS signal for different length peptides.