Dissertations, Theses, and Capstone Projects

Date of Degree

9-2023

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Yujia Xu

Committee Members

Alessandra Carriero

Lesley Davenport

Akira Kawamura

Ronald Koder

Keywords

Collagen, Collagen-Mimetic Peptides, Coiled coil, Osteogenesis Imperfecta, Gly mutations, Protein design.

Abstract

Collagen, the most abundant protein in the human body, serves as the scaffold of connective tissue and has biomaterial applications to promote wound healing. The need to study collagen further stems from a disease perspective, as mutations in collagen lead to numerous connective tissue diseases. While collagen has been explored extensively at the triple helix level, much remains undiscovered regarding its function at the fibril level due to the lack of an effective collagen model system. Using tandemly repeating “sequence units” that contain sequences from native collagen, our lab has previously developed fibril-forming collagen-mimetic peptides (FCMPs), which provide tools to study both collagen and collagenopathies. Akin to the 67 nm axial D-period of native collagen, these FCMPs form unit-staggered, 35-nm “d”-periodic fibrils.

For my thesis, firstly I developed the peptide construct U2-Nk to explore design strategies to advance these FCMPs by replacing the existing bacteriophage nucleation domain foldon with a coiled coil domain endogenous to humans—the neck region of lung surfactant protein D (SP-Nk). The findings show that U2-Nk forms d-periodic fibrils growing 2-3 µm long, further corroborating the unit-staggered model of fibrillogenesis. The findings additionally elucidate the quintessential, collaborative roles of both the nucleation domain and N-terminal Cystine knot towards its triple helical folding.

Secondly, I examined the effects of a disease-causing Gly-to-Val substitution mutation on fibrillogenesis by developing two mutant constructs of U2-Nk containing this mutation at each sequence unit to explore any resulting severity arising from differential proximity to the C-terminus. This study shows that both mutants fold into triple helices but lack the ability to form fibrils. The findings of this research work may contribute to understanding the effects of disease mutations on collagen fibrils and possible etiology of collagenopathies.

Finally, in hopes of further developing another FCMP model to study collagenopathies, I designed the peptide construct U2-877 containing two sequence units, a modified version of a previous FCMP containing three sequence units. U2-877 comprised of a continuous Hyp-free region from native collagen containing disease-causing mutations; its Hyp-free sequence was conducive for modeling in our prokaryotic expression system. Surprisingly, no d-periodic fibrils were observed for U2-877, indicating that more remains uncovered regarding the molecular mechanisms of our FCMPs fibril assembly. I also characterized the fusion U2-877 to explore whether the fusion expression tag acts similarly to the C-propeptide in its contingent removal for functional fibril assembly. Characterization of fusion U2-877 indicates that the expression tag does not prevent triple helix folding of FCMPs; whether it also prevents fibril assembly remains inconclusive due to the lack of fibrils of U2-877.

Beneficial implications of this research include insight into the etiology of collagenopathies, structure-function relationship of fibrillar collagen, and the development of biomaterials.

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