Date of Award

2024

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Globins are a protein superfamily found throughout many different organisms that are most commonly known as oxygen binding proteins, such as hemoglobin and myoglobin. Globin proteins in Caenorhabditis elegans were discovered over a decade ago, but only a few of these 33 globin proteins have been characterized. Three globin proteins, GLB-1, GLB-6, and GLB-26, have been characterized with UV-Vis, Raman spectroscopy and X-ray crystallography (Geuens, E. et al., 2010; Yoon, J. et al., 2010). In order to contribute to the limited body of knowledge about C. elegans globin proteins, we examined the heme absorbance and oxygen binding capabilities of globin proteins GLB-10, GLB-21, and GLB-31 under oxygenated and deoxygenated conditions using UV-Vis spectroscopy and GLB-1 as a control. Each protein was overexpressed in E. coli and purified. Soret and Q bands were noted in the UV-Vis spectra, because they are characteristic of the heme group. In particular, the Soret and Q bands represent the conjugated nature of the heme group and indicate the resulting electron configuration. Under oxygenated conditions, GLB-1 showed the expected spectrum with a Soret band at 414 nm, and Q bands at 543 nm and at 578 nm. GLB-10 showed a similar spectrum with a Soret band at 418 nm and Q bands at 546.8 nm and 574 nm. GLB-31 also exhibited these peaks at slightly different wavelengths, with a Soret band at 418 nm and Q bands at 540 nm and 570 nm. GLB-21 precipitated during purification, and as such, was not analyzed spectroscopically. Sodium hydrosulfite and a mixture of dithiothreitol and horseradish peroxidase were both tested as potential deoxygenation methods but were found to have little effect. Potassium ferricyanide was used to produce the met-heme spectrum of GLB-1, which showed an intense band with a peak at 420.4 nm and a weak band with a peak at 542 nm. Structure and sequence alignments were also performed using ChimeraX’s Matchmaker tool. Structurally, GLB-31 was the closest to GLB-1 overall, with a root mean square deviation (RMSD) of 4.504 Å. However, the sequence alignment showed that GLB-21 had the most conserved residues from the heme residues of GLB-1, indicating that GLB-21 has heme-coordinated residues that are the closest to GLB-1 out of all the experimental proteins. GLB-10 was the closest to GLB-6 structurally overall, with an RMSD of 3.813 Å. None of the experimental proteins had more than one heme residue that was conserved from the GLB-6 alignment, and every residue that was conserved was the predicted heme histidine of the experimental proteins. As such, it was concluded that the experimental proteins are likely pentacoordinated proteins that have different functions.

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