Journal
MOLECULAR MEMBRANE BIOLOGY
Volume 25, Issue 1, Pages 14-22Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/09687680701494007
Keywords
nuclear magnetic resonance; magic-angle spinning; quadrupolar splitting; Cdc42; Rac1; TCL
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Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 ((KKRKRK)-K-183), TCL ((KKKKKR)-K-198) and Cdc42 ((PKKSRR)-K-182) were prepared with hexalysine (K-6) and hexaarginine (R-6) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d(4)) using H-2 and P-31 NMR. The membranes retained their lamellar architecture after peptide binding, but the H-2 NMR line shapes for DMPC-d(4) indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K-6, although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R-6. Magic-angle spinning P-31 NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R-6 and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.
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