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Structural and Functional Studies of Giant Proteins in Lactobacillus kunkeei
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. (Selmer lab)
2019 (English)Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop.

In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.

Place, publisher, year, edition, pages
2019. , p. 62
Series
UPTEC X ; 19 005
Keywords [en]
structural biology, protein expression, Lactobacillus kunkeei, L. kunkeei, small angle X-ray scattering, SAXS, circular dichroism, CD, thermofluor, nano differential scanning fluorimetry, nanoDSF, honey bee, honey crop, extracellular protein
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-391664OAI: oai:DiVA.org:uu-391664DiVA, id: diva2:1345482
Educational program
Molecular Biotechnology Engineering Programme
Supervisors
Examiners
Available from: 2019-08-29 Created: 2019-08-25 Last updated: 2019-08-29Bibliographically approved

Open Access in DiVA

The full text will be freely available from 2022-08-25 16:25
Available from 2022-08-25 16:25

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