Abstract
The interaction of the complementary K (Ac-(KIAALKE)3-GW-NH2) and E (Ac-(EIAALEK)3-GY-NH2) peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009) is investigated by electron paramagnetic resonance (EPR). By frozen solution continuous-wave EPR and double electron-electron resonance (DEER), the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm) and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.
Highlights
All living organisms utilize membrane fusion for their normal functioning
To gain a better picture on membrane fusion, we focus on the coiled-coil zipper segment of the complex, which consists of the helical peptides K and E, for sequences see Table 1
The cw-electron paramagnetic resonance (EPR) spectra (Fig 3a) of the samples are shown in red and are superimposed on the cwEPR spectrum of MTSL measured under the same conditions as a monomeric reference
Summary
Cellular activities that involve membrane fusion are hormone secretion, enzyme release, neurotransmission etc. Membrane fusion needs a specialized set of proteins, such as the SNARE protein complex [1,2,3,4,5,6,7] (SNARE: soluble NSF attachment protein receptor; NSF = N-ethylmaleimide-sensitive factor). Membrane fusion induced by SNARE involves the coiled-coil interaction between three complementary SNARE proteins [8,9]. To understand protein-mediated membrane fusion, a coiled-coil model system mimicking the complex of SNARE proteins was designed [10,11,12,13].
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