Solid State NMR Structural Analysis of the RADA16-I Designer Self-Assembling Peptide

• Other Authors: Cormier, Ashley (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemical engineering; Biomedical engineering
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-6900

The main product of this work is the detailed molecular structure of nanofibers formed by the RADA16-I designer peptide. RADA16-I is a designer self-assembling peptide that has shown great utility in the area of tissue engineering. RADA16-I may be a promising new material for regenerative medicine, drug delivery, and cell culture; however, little is known on how or why this peptide self-assembles or exhibits useful properties. Useful properties of RADA16-I include its ability to dynamically reassemble (self-heal) after fragmentation (e.g. mechanical stress) without the need for added monomer. RADA16-I also forms a three-dimensional scaffolding system that could potentially provide a structure for cells to proliferate and grow that mimics the extracellular matrix. In order to understand more about this peptide, solid state nuclear magnetic resonance (NMR) experiments were combined with other techniques in order to fully characterize the molecular (secondary, tertiary, and quaternary) structure of RADA16-I self-assembled nanofibers. We developed a full atomic level structural model for RADA16-I nanofibers self-assembled in solution using solid state NMR measurements on selective isotopically labeled samples. Before the present studies, RADA16-I was thought to form an antiparallel β-sheet structure. However, by using a combination of solid state NMR, simulations, and molecular modeling, RADA16-I was reasoned to have a parallel β-sheet structure that has a registry shift of 2 residues between adjacent β-strands in the same β-sheet. Solid state NMR experiments were performed on various samples labeled with13C at key places directed through molecular modeling. NMR peak positions and linewidths indicate an ordered structure composed of β-strands. Through NMR analysis we were able to conclude that the nanofibers are composed of two stacked β-sheets stabilized by a hydrophobic core formed by alanine sidechains, consistent with previous proposals. However, the previously proposed antiparallelβ-sheet structure is ruled out by13C-13C dipolar couplings. Instead, neighboring β-strands within β-sheets are parallel, with a registry shift that allows for cross-strand staggering of oppositely charged arginine and aspartate sidechains. The resulting structural model has nanofiber dimensions that are consistent with images taken by transmission electron microscopy and atomic force microscopy. Multiple NMR peaks for each alanine sidechain were observed and can be attributed to multiple configurations of the charged sidechains on the nanofiber surface. In the process of synthesizing this peptide and self-assembling it to form nanofibers, we found RADA16-I to be difficult to dissolve. Low solubility presented a problem for nanofiber structural characterization due to low yields of nanofiber samples. Our efforts to understand this problem occurred before the analysis described in Chapter 2. We hypothesized that this loss of solubility was caused by RADA16-I forming a stable structure in the solid state which prevents solubility. This solubility issue was further analyzed by studying the structure of RADA16-I in dry powder form. These efforts were made in order to make the process of producing RADA16-I nanofibers more efficient. RADA16-I was found to self-assemble in the solid state from an α-helical structure into an organized β-sheet structure upon heating or time at room temperature. This structural conversion was found to be correlated with a decreased amount of solubility. Water (1 mL / 1 mg peptide) appeared to enhance the peptides ability to convert to β-strand secondary structure and assemble into β-sheets. However, temperature dependent Fourier transform infrared spectroscopy and time dependent wide-angle X-ray diffraction data indicate that bound water from the atmosphere may hinder the assembly of β-strands into β-sheets. We suggest that secondary structural transformation and intermolecular organization together produce a water-insoluble state. These results reveal insights into the role of water in self-assembly of polypeptides with hydrophilic sidechains, and have implications on optimization of RADA16-I nanofiber production. Solid state NMR measurements on selective 13C-labeled RADA16-I peptide were used to determine whether or not β-sheets formed through undesirable solid state self-assembly in synthetic RADA16-I peptide had the same structure as β-sheets found in RADA16-I nanofibers. Isotopic labeling on the methyl carbon of the fourth amino acid, alanine, (A4 C β) allowed production of samples with systematic variation ofα-helix and β-strand content as well as measurements of inter-molecular13C-13C nuclear dipolar couplings with the PITHIRDS-CT NMR experiments. This structural transition was also characterized by Fourier transform infrared spectroscopy (FTIR) and wide angle X-ray diffraction (WAXD). Independence of PITHIRDS-CT decay shapes on overall α-helical and β-strand content suggest a nucleation-dependent conversion of α-helices to organized β-sheets because there is no evidence that β-strands exist without being incorporated into β-sheets. RADA16-I in solid state was found to have a different structure than that of the solution self-assembled RADA16-I that forms nanofibers. Even water addition to RADA16-I in the solid state led to a structure that did not match the structure of the RADA16-I nanofibers. We have concluded that the difference between the RADA16-I in solid form, or even with water addition, to the RADA16-I that has been self-assembled into nanofibers is that the RADA16-I in dry form is a single in-register parallel β-sheet where the nanofiber form is stacked β-sheets. === A Dissertation submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Fall Semester, 2012. === October 29, 2012. === nanofibers, RADA16-I, Self-assembly, solid state NMR, structure === Includes bibliographical references. === Anant Paravastu, Professor Directing Dissertation; Geoffrey Strouse, University Representative; Samuel Grant, Committee Member; Jingjiao Guan, Committee Member.