Association of a model transmembrane peptide containing
Gly in a heptad sequence motif
James D. Lear 1*, Amanda Stouffer 1, Holly Gratkowski 2, Vikas Nanda 1 and William F. DeGrado 1
1 University of Pennsylvania
2 IBM Life Sciences
* To whom correspondence should be addressed. E-mail: lear{at}mail.med.upenn.edu.
Submitted on August 28, 2003
Revised on November 19, 2003
Accepted on 27 July 2004
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Abstract |
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A peptide containing glycine at "a" and "d" positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A (GpA) transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1, 3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the AUC-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by AUC, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif
described for GpA.
Key Words:
FRET, analytical ultracentrifugation, antiparallel quenching model, coiled-coil, membrane peptides
