Applications in Peptide Design: Self-assembly of Collagen Triple Helices and β-sheet Nanofibers

Jeffrey Hartgerink - Rice University

Nature uses self-assembly to generate nearly every complex structure from the DNA double helix to the lipid membrane to the intricate folding of the ribosome; a huge number of individually weak noncovalent interactions are used to direct the assembly of life’s machinery. This approach allows for easy reversibility and self-healing properties that are commonplace in nature but difficult to engineer. Our lab has focused on mimicking nature’s approach to the preparation of nanostructured materials through the design of peptides. In this talk I will describe our work with Collagen Mimetic Peptides (CMPs) which selfassemble into triple helices and MultiDomain Peptides (MDPs) which self-assemble into b-sheet nanofibers.

CMPs differ in critical ways from other well studied proteins in that they lack a hydrophobic core and require a glycine every third amino acid creating a characteristic (Xaa-Yaa-Gly)n repeat. Design of synthetic triple helices which can mimic collagen are extremely challenging for many reasons, but one of the most apparent is that a system of three peptides A, B and C can self-assemble into at least 27 different compositions and registers (AAA, AAB, AAC, ABA, etc). Proper design therefore must stabilize one of these while destabilizing the other 26. I will describe the design, synthesis and characterization of several AAB and ABC heterotrimers and computational methods which allow us to both predict their stability and allow rapid and accurate design of new triple helical systems. An additional challenge in the application of collagen triple helices is their extremely slow rate of folding and relatively poor equilibrium between monomer and trimer. To overcome these limitations we have developed a method of covalent capture that perfectly preserves the three dimensional architecture of the helix while simultaneously improving their thermal stability and eliminating equilibrium to the monomer. With these advances we are now working towards biomaterial applications that allow selective targeting of collagen specific integrins in a robust fashion not previously possible.

MDPs self-assemble into nanofibers approximately 8 nanometers wide and many microns in length through the interplay of several non-covalent interactions including hydrogen bonding and the hydrophobic effect, which drive assembly, and ionic repulsion which can act as a controlling switch to turn assembly on or off. As nanofibers grow, they entangle with one another and, in water, will form a viscoelastic hydrogel at concentrations at or above 0.5% by weight. These hydrogels can be loaded with small molecule drugs, proteins, cells or a combination of all these to achieve syringe directed deliver with control over release kinetics. However, the nanofibrous hydrogel can also be used unloaded where we have found that, free of any bioactive agents, it is rapidly infiltrated with cells in vivo and remodeled into highly vascularized tissue. I will discuss applications of MDPs in wound healing, nerve regeneration and cancer immunotherapy.

 

 

Collagen Mimetic Peptide and MultiDomain Peptide structure. a) Repetitious (Xaa-Yaa-Gly)n sequence required for collagen triple helix assembly. b) Model of a collagen triple helix. c & d) Highlight of the 45 membered ring formed upon collagen covalent capture. e) Example sequence of an MDP. f) Scheme of MDP self-assembly.

 

Reception following – Louderman 561