Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Raich, Andy

Committee Member/Reader

Harriss, Edmund

Committee Member/Second Reader

Sakon, Joshua

Abstract

In order to spread infections, bacterial collagenases methodically unravel collagen fibril in tissues. Collagen is the most abundant protein in the body, and can be found in the skin, bone and cartilage [1]. Two collagenases, ColG and ColH, synergistically dismantle collagen fibrils by seeking different weak links in the collagen structure. The collagen-binding domain (CBD) of these collagenases binds to most vulnerable regions in collagen [8]. Without CBDs, collagen fibril cannot be degraded. Cells express collagen receptors in order to anchor themselves, which is a critical step in cell proliferation. Binding sites for some collagen receptors, such as integrin and interleukin-2, have been determined [4]. The goal of my project is to determine where tandem CBD from ColG binds on rattus type I collagen. Out of the more than 20 different types of collagen in the human body, this type of collagen is the most abundant [4]. Biophysical studies using collagen-like peptides have demonstrated that the CBD targets less tightly wound (undertwisted) regions in the triple-helix [8]. Therefore, it is expected that the CBD also targets proline poor regions in collagen. It is also expected that the CBD does not interfere with binding of important anchoring proteins based on previous studies of ColH. In order to determine whether these hypotheses are correct, my approach is to use a photo-reactive crosslinker to freeze the CBD-collagen complex [16]. The crosslinker has a sulfhydryl reactive functional group. Adjacent to the collagen-binding cleft in CBD, a cysteine mutation was introduced by means of an overlap extension polymerase chain reaction (PCR). This cysteine mutation allows for the crosslinker to be attached by forming a disulfide bridge. After performing the PCR reactions, the mutant proteins were successfully overexpressed and purified. Following purification, several crosslinkers were attached to the cysteine residue, and the results were confirmed using mass spectrometry. These attached photoreactive crosslinkers consists of a photo-reactive functional group. After UV light exposure the cross-linker should be activated to attach to the collagen forming a CBD-collagen complex. Future experiments then subjected the complex to digestion by trypsin, and the tryptic fragments were analyzed by LC-MS/MS (ESI mass spectrometry) to identify the location of CBD binding. Fusion proteins of ColH CBD and signal molecules, such as cytokines, hormones and growth factors, anchor themselves to rapidly remodeling collagen [15]. This anchoring promotes tissue engineering and wound repairs in vivo [12, 13]. It also reduces the amount of signal molecules required to achieve the desirable outcome while at the same time reducing side effects. Although the tandem CBD from ColG is safe in vitro, fusion proteins of the tandem CBD and signal molecules have not yet been made and tested in vivo. My findings may contribute to the continuum of research to lead to efficacious treatment options for wound repair and tissue engineering applications.

Keywords

Collagen fibril, Collagen-binding domain, Molecular, CBD

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