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 drug delivery and medical applications. C-D labeled valine was synthetically introduced into five distinct positions of the twenty-residue MAX1 ß-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.
ABSTRACT
Precise control over self-assembly of peptides into adaptable nanostructures allows for emulating the dynamic organization of natural proteins and their sophisticated biological functions. Utilization of disease biomarkers as internal stimuli for manipulating the self-assembly of peptides bestows their adaptable features with a spatiotemporal resolution to satisfy the requirement of the performance of biomaterials. Reactive oxygen species (ROS) consisting of reactive ions or free radicals are overexpressed by pathological lesions and have been recognized as one of conventional biomarkers for disease progression. Despite the progress made over the past decade in stimulus-responsive self-assembly of peptides as well as the summarization of this progress, the specific reviews focusing on ROS-responsive self-assembly of peptides remain scarce. This review summarizes the progress achieved over the past decade of the ROS-responsive self-assembly of peptides into adaptable nanostructures and their applications in biomaterials. We focus on the chemical sources responsible for the ROS-sensitive behavior of peptides, in which the chemical moieties are incorporated into peptides as side chains, terminal groups, or backbone linkages. The ROS-responsive self-assembly of peptides into nanostructures with morphologies adaptable to ROS-oxidation and their applications in enzymatic catalysis, chemosensing, drug delivery, and tissue engineering will be highlighted. Understanding the established ROS-responsive peptide self-assembling systems allows us to provide perspectives for their further development and thereby elucidate their great potential in development of advanced biomaterials.
