Direct observation of peptide hydrogel self-assembly

• Published in: Chemical science (Cambridge) Vol. 13; no. 34; pp. 12 - 128
• Main Authors: Adams, Zoë C, Olson, Erika J, Lopez-Silva, Tania L, Lian, Zhengwen, Kim, Audrey Y, Holcomb, Matthew, Zimmermann, Jörg, Adhikary, Ramkrishna, Dawson, Philip E
• Format: Journal Article
• Language: English
• Published: CAMBRIDGE Royal Soc Chemistry 31.08.2022; Royal Society of Chemistry; The Royal Society of Chemistry
• Subjects: Absorption spectra; Biocompatibility; Biomedical materials; Chains; Chemistry; Chemistry, Multidisciplinary; Deuterium; Enzyme kinetics; Hydrogels; Infrared spectroscopy; Peptides; Phase separation; Physical Sciences; Proteins; Science & Technology; Self-assembly; Solubility; Spectrum analysis; Steady state models; Structural models; Valine; AMYLOID FORMATION; CONFORMATIONAL HETEROGENEITY; PROTEIN-STRUCTURE; SIDE-CHAIN; KINETICS; 2D IR SPECTROSCOPY; CARBON-DEUTERIUM BONDS; INFRARED-SPECTROSCOPY; BETA-HAIRPIN PEPTIDE; PROBE
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d1sc06562a

The characterization of self-assembling molecules presents significant experimental challenges, especially when associated with phase separation or precipitation. Transparent window infrared (IR) spectroscopy leverages site-specific probes that absorb in the "transparent window" region of the biomolecular IR spectrum. Carbon-deuterium (C-D) bonds are especially compelling transparent window probes since they are non-perturbative, can be readily introduced site selectively into peptides and proteins, and their stretch frequencies are sensitive to changes in the local molecular environment. Importantly, IR spectroscopy can be applied to a wide range of molecular samples regardless of solubility or physical state, making it an ideal technique for addressing the solubility challenges presented by self-assembling molecules. Here, we present the first continuous observation of transparent window probes following stopped-flow initiation. To demonstrate utility in a self-assembling system, we selected the MAX1 peptide hydrogel, a biocompatible material that has significant promise for use in drug delivery and medical applications. C-D labeled valine was synthetically introduced into five distinct positions of the twenty-residue MAX1 β-hairpin peptide. Consistent with current structural models, steady-state IR absorption frequencies and linewidths of C-D bonds at all labeled positions indicate that these side chains occupy a hydrophobic region of the hydrogel and that the motion of side chains located in the middle of the hairpin is more restricted than those located on the hairpin ends. Following a rapid change in ionic strength to initiate self-assembly, the peptide absorption spectra were monitored as function of time, allowing determination of site-specific time constants. We find that within the experimental resolution, MAX1 self-assembly occurs as a cooperative process. These studies suggest that stopped-flow transparent window FTIR can be extended to other time-resolved applications, such as protein folding and enzyme kinetics. To facilitate the characterization of phase-transitioning molecules, site-specific non-perturbative infrared probes are leveraged for continuous observation of the self-assembly of fibrils in a peptide hydrogel following stopped-flow initiation.