Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins.
We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(±50) nM, corresponding to a free energy of dissociation of 9.0(±0.1) kcal mol−1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd = 1.7(±0.2) μM and 4.2(±0.9) μM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.
Keywords
two-stage model
membrane protein
glycophorin A
ultracentrifugation
C8E5
Abbreviations
C8E5
pentaoxyethylene octyl ether
C12E8
octyloxyethylene dodecyl ether
SN
staphylococcal nuclease
GpA
glycophorin A
SN/GpATM
the chimeric protein of SN and the transmembrane domain of GpA
SN/GpATM(L75A)
SN/GpATM with leucine at GpA position 75 mutated to alanine
SN/GpATM(I76A)
SN/GpATM with isoleucine at GpA position 76 mutated to alanine