LMCA1 as prototype system for the study of Ca2+ pumps in nanodisks

• Main Author: Magkakis, Konstantinos
• Format: Others
• Language: English
• Published: Umeå universitet, Kemiska institutionen 2021
• Subjects: Chemical Sciences; Kemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-185547

Membrane proteins make up approximately a third of all proteins and serve as prime drug targets, yet their structural and biophysical characterization presents some unique challenges due to their hydrophobic surfaces and flexible structure. The main goal of this project was to compare, with the use of activity assays, two methods employed for the experimental handling of membrane proteins. The methods compared were the traditional method of using detergent micelles to keep the membrane proteins in solution, with the novel technique of reconstituting the proteins in nanodisks, using the LMCA1 P-type ATPase as a prototype. The secondary goal was to successfully express, purify and reconstitute in nanodisks, an inactive mutant of the LMCA1 protein to use as a control for the activity measurements. As of the end of this project the inactive LMCA1 mutant was successfully reconstituted into MSP1D1 nanodisks and the initial assay results showed higher activity for the reconstituted protein. This points to the conclusion that nanodisks could provide an environment that is more native for the protein, compared to detergent micelles, which can lead to greater precision in their structural and biophysical characterization.