Structural Stability of Human Prosecretory Mitogen Lacritin
Channel: JMU ISAT
Date: 2017-07-01

Full Title: Analysis of Structural Stability of Human Prosecretory Mitogen Lacritin by Circular Dichroism by Annie Desmarais and Casey Cortes
Advised by: Dr. Robert McKown
Abstract:
Purpose:
Lacritin is a human tear glycoprotein that has high thermal stability. Biological activities reside in 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 is to investigate the hypothesis that the salt bridges within the alpha helices contribute to the high thermal stability.

Methods:
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. Protein concentrations and purity were determined by BCA assay and SDS PAGE, respectively. 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 two controls, Bovine Serum Albumin (BSA) and lysozyme, and unaltered lacritin. Data collected was analyzed with an alpha helix formula to determine the percent alpha helix structure at ten degree increments from 25- 85°C, 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 exhibited significant decrease in alpha helical structure while the alpha helical structure of normal lacritin increased slightly. At 25°C the mutants had 12-25% less alpha helix than unaltered lacritin and 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. This was shown by reduced percent alpha helix at 25°C for all salt bridge mutants and no significant structural change once temperature was increased.