Investigation of the assembly mechanism and molecularlevel structure of β-hairpin peptide hydrogelators
Date
2015
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Designed and naturally derived self-assembling peptides have utility as
building blocks in the fabrication of hydrogels as tissue engineering platforms and
drug delivery depots. Additionally, designed peptides and those derived from
amyloidogenic proteins can serve as tools to understand the biophysical principles that
guide amyloid-like fibril formation and define the molecular-level interactions that
constitutes the assembled state. Similarly to amyloid-derived self-assemblers, many
designed material-forming peptides also assemble to form -sheet rich fibrils. To
date, however, mechanistic and structural determination of biomaterial-based peptide
assemblies significantly lags behind efforts to define assemblies of disease-associated
amyloid fibrils. To this end, the mechanism of assembly and final solid state structure
of the self-assembling hydrogelator MAX1 has been investigated in this dissertation.
MAX1 is a 20-amino acid peptide designed de novo to form a facially amphiphilic -
hairpin. The peptide is composed of two strands of alternating hydrophobic valine and
hydrophilic lysine residues that flank a four residue turn-promoting sequence (-
VDPLPT-). Under aqueous, low ionic strength conditions, the peptide adopts an
ensemble of random coil conformations due to electrostatic repulsion between lysine
side chains. Upon addition of a high pH or high ionic strength solution, the lysine
borne charge is attenuated and the peptide assembles to form a -sheet rich network of
fibrils that constitute a self-supporting hydrogel. The objective of this work is to identify the mode of assembly and the final
molecular-level structure of the MAX1 peptide alone and in the presence of its
enantiomer DMAX1. For pure solutions of MAX1, it was found that random coil,
monomeric peptide undergo hydrophobic collapse to form unstructured oligomers that
precede the formation of -sheet structure. The data strongly argue against an initial
unimolecular folding event that subsequently leads to fibril formation. The early time
assembly events are defined by a multi-component solution of peptide composed of
both unstructured oligomers and monomeric peptide. Overtime, these species become
competent for the formation of the -sheet rich fibrils that define the final assembled
state. The molecular-level structure of MAX1 within these peptide fibrils are further
investigated by solid state NMR. It is shown that MAX1 forms the designed -hairpin
secondary structure with a type II’ -turn. Within fibrils, hairpins are arranged in a
Syn/Anti configuration where turns are aligned along a given -sheet monolayer but do
not directly oppose each other across the fibril bilayer. This arrangement leads to the
formation of critical hydrophobic and van der Waals interactions that are not
accessible in completing arrangements and, thus, leads to the formation of highly
monomorphic fibrils within a kinetically trapped network. In separate work, the
molecular-level interactions between peptide enantiomers MAX1 and DMAX1 in coassembled
fibrils are investigated. Hydrogels formed from enantiomeric mixtures of
MAX1 and DMAX1 are significantly more rigid than hydrogels produced from either
peptide alone, with the racemic mixture exhibiting a four-fold increase in mechanical
rigidity. Using an arsenal of rheological, spectroscopic, scattering, and microscopic
techniques it was determined that the peptide enantiomers co-assemble to form fibrils
that are more stiff than those formed by either pure MAX1 or DMAX1. The fibril
stiffness stems from key nested hydrophobic interactions between laterally associated
enantiomers that cannot be accessed within pure enantiomeric assemblies and is the
ultimate reason for the enhanced mechanical rigidity of the racemic hydrogel. Finally,
in a separate project, hydrogels formed from negatively charged -hairpins are
explored as drug delivery vehicles. Here, the effect of network electrostatics on the
release of a series of model proteins is investigated. The data show that attractive
electrostatic interactions play a large role in the retention of proteins within the
hydrogel network and open the door to the further optimization of this new class of
gels. Overall, this thesis demonstrates how knowledge of the assembly mechanism
and final molecular structure of naturally-occurring and disease-related self-assembled
peptides can be applied to material formation peptides and further lead to the rational
optimization of peptide assembly and ultimate biomaterial mechanical behavior.