Direct Observation of Peptide Hydrogel Self-Assembly

16 December 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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 tissue regeneration and drug delivery. 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 gelation, 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 gelation 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.

Keywords

peptide
hydrogel
FT-IR
deuterium
kinetics
self-assembly

Supplementary materials

Title
Description
Actions
Title
Supporting Information for Direct Observation of Peptide Hydrogel Self-Assembly
Description
materials, circular dichroism spectra, mass spectra
Actions

Supplementary weblinks

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