Development of Collagen Mimetic Mini-Fibrils with Tunable Collagenase Susceptibility

<p>Collagen fibrils are the primary molecular scaffold of all connective tissues and provide the essential support for cell migration and differentiation. Collagen fibrils represent one of the most complex molecular hierarchies: three polypeptide chains in Gly-Xaa-Yaa repeating amino acid sequences, first form the rod-shaped collagen triple helix. These triple helices further self-assemble laterally in a specific manner to form fibrils having the characteristic 67 nm axially repeating structure known as the D-period. Enzymatic digestion of collagen fibrils by matrix metalloproteinases (MMPs) is critical for several physiological processes and is of particular consequence during wound healing. Many of the mechanisms of function of MMPs on collagen fibrils are not completely understood. The focus of my thesis is to use protein engineered, collagenmimetic fibrils (CMF) to investigate molecular mechanisms of MMP and to explore the potential of these CMFs as biomaterials offering tunable susceptibility to MMPs for wound healing and other biomedical applications.</p> <p>To achieve this goal, I first studied the interactions of MMP1 and the bacterial collagenase ColG with native type I collagen. Kinetic assays of the enzyme reactions revealed that the triple helix was the most susceptible conformation of collagen to MMP1, while the degradation of the fibrillar form or the unfolded form of collagen was significantly slower. The ColG, on the other hand, is very effective at cleaving collagen in its unfolded form. The action of ColG is impeded by the higher order structures of the triple helix and the fibrils, although the tight packing of the triple helices in the fibrils does not seem to offer more protection against the attack of ColG than the triple helical conformation per se.</p> <p>The major effort of my research was devoted to studying the MMP1 susceptibility of three designed CMF peptides. The CMFs are produced by bacterial expression using designed genes. The amino acid sequences of the CMF are selected to model the section surrounding the MMP1 digestion site of type III collagen. Type III collagen is one of the major fibrillar collagens of skin and is critically involved in the early stages of wound healing. All three peptides formed stable triple helix with a melting temperature of ~42 °C. The triple helices further self-assemble to form mini-fibrils having D-period-like axially repeating structures. The fibrillogenesis of the minifibrils showed similar dependence on the temperature, the ionic strength, and the pH to those of native collagen fibrils, indicating the mini-fibrils are stabilized by similar molecular interaction as those in native collagen fibrils. The three peptides, colt3_0, colt3_1, and colt3_2, were designed to have, respectively, none, one, and two MMP1 digestion sites. The kinetic assays of MMP1 with the three mini-fibrils demonstrated that the colt3_0 is highly resistant to MMP1, while colt3_2, having two MMP1 digestion sites, is the most susceptible. Unfortunately, the low yields of the peptides prevented more detailed studies on the mechanisms of MMP1 with the CMFs as the substrate. The research, nonetheless, demonstrated that the mini-fibrils are good models for collagen fibrils and offer a unique opportunity to investigate the molecular interactions of MMP1 with collagen.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biochemistry

Grantor

The Graduate School and University Center of The City University of New York

Year dc:date.available

2024

Author dc:creator

• Chaugule, Jui

Advisor dc:contributor.advisor

• Yujia Xu

Committee members dc:contributor.committeemember

• Nancy Greenbaum
• Edward Kennelly
• James SanAntonio
• Madan Balaraman

• Biochemistry
• Biotechnology
• MMP1 Collagen biomaterial

Repository record dc:identifier

https://academicworks.cuny.edu/gc_etds/5985

OAI identifier oai:identifier

oai:academicworks.cuny.edu:gc_etds-7106