Characterizing membrane proteins using biophysical techniques remains a challenge due to maintaining the structural integrity of the membrane protein in vitro. Recently, a novel membrane mimetic system has come of age where an amphiphilic copolymer, styrene-maleic acid (SMA), is added to heterogeneous lipid bilayers to form small homogeneous discs (~10 nm) called styrene-maleic acid copolymer lipid nanoparticles (SMALPs).1 SMALPs are advantageous because it provides a homogeneous lipid bilayer environment for easy reconstitution of the membrane protein without the use of detergent for membrane protein reconstitution. However, there are limitations, such that SMA will crash out at a pH lower than 6.5 and under high amounts of divalent cations such as Mg2+.2 Recently, polymer chemists in our research team are one of only a few labs that have pioneered alternative synthesis of SMA by synthesizing SMA with various functional groups, such as an amino ethanol (SMA-AE), glucosamine (SMA-Glu) to mimic lipid head groups, neutral charged polymers (SMA-Neut), and positively charged groups (SMA-Pos).3
KCNE1 is a 129 amino acid, single transmembrane protein that assists in the proper functioning of several voltage-gated potassium ion channels. Mutations in this protein cause significant damage to humans, as it can result in congenital deafness, congenital Long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden infant death.
In this study, site-directed mutagenesis of KCNE1 mutants were made, overexpressed, purified, spin labeled, and reconstituted into various nanodisc systems. CW-EPR was used to probe how the various membranes influenced membrane protein dynamics. Critical analysis will reveal which membrane mimetic system provides the most native-environmen
Authors: Benjamin D. Harding
Faculty Advisors: Kevin M. Burridge, Rebecca Stowe, Indra D. Sahu, Carole Dabney-Smith, Richard E. Edelmann, Dominik Konkolewicz, Gary A. Lorigan, Department of Chemistry and Biochemistry
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