Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Date of Award
Bachelor of Science (BS)
Department of Integrated Science and Technology
Purpose: Lacritin is a human tear glycoprotein that has high thermal stability. When cleaved, lacritin has antimicrobial activity resulting from the C-terminus amphipathic alpha helical region. The alpha helices contain three salt bridges; ionic bonds between neighboring oppositely charged amino acids. The purpose of this research was to investigate the hypothesis that the salt bridges within the alpha helices contribute to the high thermal stability.
Methods: To determine the role of salt bridges in the thermal stability of lacritin, point mutants were prepared for each salt bridge by site directed mutagenesis that replaced the oppositely charged amino acids with serine. The point mutants were expressed in E. coli and purified. Western blot analysis confirmed the identity of lacritin proteins. Circular dichroism (CD) was used to study conformational changes in the secondary structure of these mutants compared to unaltered lacritin along with two controls, bovine serum albumin (BSA) and lysozyme. Data collected was analyzed with the alpha helix formula to determine the percent alpha helix structure at ten degree increments from 25-85⁰C, using poly-l-lysine as the standard.
Results: The mutated proteins reacted with lacritin specific antibodies in Western blot analysis. Under thermal denaturation conditions, the control proteins both had a significant decrease in alpha helical structure while alpha helical structure of normal lacritin increased slightly. At 25⁰C, the mutants had 12-25% less alpha helix than unaltered lacritin. Increasing the temperature did not have a significant impact on alpha helix structure.
Conclusions: The salt bridges play a role in formation of the alpha helices but not in overall thermal stability of lacritin.
Ramirez Cortes, Casey Q. and Desmarais, Anna P., "Analysis of structural stability of human prosecretory mitogenic lacritin by circular dichroism" (2017). Senior Honors Projects, 2010-current. 320.