Zoë Adams (Scripps Research)| Erika Olson (Scripps Research)| Zhengwen Lian (Scripps Research)| Matthew Holcomb (Scripps Research)| Ramkrishna Adhikary (Scripps Research)| Jörg Zimmermann (Scripps Research)| Philip Dawson (Scripps Research)
The physical characterization of molecules that self-assemble presents a challenge, particularly when associated with phase separation or precipitation. Infrared (IR) spectroscopy has been shown to be a non-perturbative method for monitoring folding dynamics, accentuated by the sensitivity of the bond stretch frequencies of site-specific IR probes to changes in local molecular environment.1 Site-specific incorporation of probes that absorb in the “transparent window” region of the biomolecular IR spectrum has been widely used to gain insight into protein electrostatics, structure, folding, and conformational dynamics. To expand the use of IR probes for the investigation of time-resolved kinetic processes, such as self-assembly, carbon-deuterium (C-D) bonds were incorporated into five different positions of a twenty-residue MAX1 β-hairpin peptide and the site-specific kinetics of gelation were examined after stopped-flow mixing using FTIR detection. MAX1 peptide hydrogels are biocompatible and clinically relevant for application in tissue regeneration and drug delivery.2 Steady state IR absorption frequencies and linewidths of C-D bonds at all labeled positions indicate, as expected, that the labeled side chains occupy a hydrophobic region of the hydrogel; furthermore, the motion of side chains located in the middle of the hairpin is more restricted than those located on the hairpin ends. The changes in the absorption spectra of C-D bonds induced by gelation were reliably followed as function of time and the site-specific time constants for the gel transition show that MAX1 gelation occurs as a cooperative process with no lag phase. We believe that stopped-flow FTIR spectroscopy is equally applicable for other transparent window IR probes and can be extended to other time-resolved applications, such as protein folding and enzyme kinetics.
1. Adhikary, R., J. Zimmermann, and F.E. Romesberg, Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution. Chem Rev, 2017. 117(3): p. 1927-1969.
2. Schneider, J.P., et al., Responsive Hydrogels from the intramolecular Folding and Self-Assembly of a Designed Peptide. J. of Am. Chem. Soc., 2002. 124: p. 15030-15037.
Gelation of MAX1 hydrogel-forming peptides can be reliably followed via the red-shift in infrared (FT-IR) peak frequency of site-specific Carbon-Deuterium bond labels, resulting in steady-state and time-resolved characterization.