Biochemical Characterization of Wild-Type Hsp27 and Point Mutation S135F That Leads to Neurodegenerative Disease

Janelly Villalobos, University of Texas at El Paso

Abstract

HSPB1, also classified as heat shock protein 27 (Hsp27), is a small chaperone that is active in cells during stressful conditions. The chaperone can stabilize target proteins in a non-aggregated folding state and might be involved in regulation of folding and assembly of neurofilaments. There are five mutations in the genome of Hsp27 that are involved in Charcot Marie Tooth Disease (CMT). This neurodegenerative disease is characterized by the first decades of life where it slowly progresses to weakness of muscles followed by sensory loss and skeletal deformities. CMT is the most common hereditary neuromuscular disease, with an estimated rate of 1 in 2500 people in the United States. It is still not clear what role the mutations play in the mechanism that leads to the disease. Some studies have shown that the mutations result in an abnormal binding affinity between the mutant and wild-type protein. This project aims to study the function and structure of the wild-type Hsp27 compared to one of the mutations S135F. The gene for Hsp27 was cloned and transformed into BL21(DE3) bacterial cells for protein expression. It was then purified using anion exchange chromatography followed by size exclusion chromatography. The chaperone protein activity in the oligomerization state was assayed by using alpha-lactalbumin aggregated by a reducing agent, dithiothreitol (DTT) in-vitro. The secondary structure of Hsp27 WT and mutation S135F were evaluated through the following biophysical analyses: circular dichroism and dynamic light scattering. The structure of mutation S135F was evaluated under a transmission electron microscope.

Subject Area

Biochemistry

Recommended Citation

Villalobos, Janelly, "Biochemical Characterization of Wild-Type Hsp27 and Point Mutation S135F That Leads to Neurodegenerative Disease" (2017). ETD Collection for University of Texas, El Paso. AAI10743227.
https://scholarworks.utep.edu/dissertations/AAI10743227

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