Lateral dynamics of proteins with polybasic domain on anionic membranes: A dynamic Monte-Carlo study Vladimir Kiselev PhD student The University of Edinburgh CSBE seminar, Edinburgh, UK 18 November 2010 

Electrostatics in protein- membrane interactions 20-40% +++++ +++++ +++++ +++++ PA, PS (-1) PIP2 (-4) 

Example: proteins with polybasic domain c-Src MARCKS Murray D. et al. Structure (London, England : 1993) 5, no. 8 (August 1997): 985-9 Gambhir A. et al. Biophysical journal 86, no. 4 (April 2004): 2188-207 

Endocytosis Arf6 (+) ARNO PIP2 (-4) PLD PA (-1) PIP5K AP-2 Gundelfinger E. et al. Nature reviews. Molecular cell biology 4, no. 2 (March 2003): 127-39 •  Spatial dimension: ~100 nm •  Time: ~1 s •  Feedback loops (↑+,↑-) •  Electrostatics (+,-) 

Lipid gradients in endocytosis PLD Zeniou-Meyer M. et al. The Journal of biological chemistry 282, no. 30 (July 2007): 21746-57 PI4P PIP2 PIP5K ATP ADP Iman van den B. and Nullin Divecha. Journal of cell science 122, no. Pt 21 (November 2009): 3837-50 Free choline Water Membrane - - - - - - - - - + + - 

Model setup d=0.8nm 

PS (-1) is weakly sequestered by the peptide PS (-1) density At 25-35% PS the peptide is enriched with PS only by 1.3 No PIP2 Weak sequestration of PS lipids on binary membranes Exp 

PIP2 (-4) is strongly sequestered by the peptide PIP2 (-4) density At 1% PIP2 the peptide is enriched with PIP2 by 10 The peptide sequesters mainly PIP2 lipids, but not PS Exp 

PIP2 drastically changes electrostatic properties of peptide On binary PC/PS membranes the peptide is always positive Already at 0.2% PIP2 the peptide becomes negative Peptide total charge 

The peptide diffuses mainly with PIP2 lipids but not with PS Average association time of lipids bound to the peptide Exp 

Presence of PIP2 reduces the peptide diffusion rate Binary Ternary Exp Exp 

What happens if there is a lipid gradient on the membrane? Peptide charge Value of gradient Peptide diffusion coefficient PLD PLD 

Simulations are in agreement with analytical estimations Relevant biological conditions: V = 3 µm/s ~ 10 µm Cell Time Displacement 

Addition of PIP2 switches the effect PIP2 

Continuous model 30% t = 0.2 s 13.3 times •  Dimension: ~100 nm •  Time: ~1 s Endocytosis 

Conclusions •  Model predicts formation of a “lipid shell” in the vicinity of the PB •  PB domain sequesters mainly PIP2 lipids, but not PA or PS •  PB diffusion rate weakly depends on undisturbed membrane composition •  In the presence of lipid gradient PB drifts in the region of higher lipid density – spontaneous clustering 

Future work 1) Extending the continuous model by including: –  protein membrane-cytoplasmic shuttling –  local lipid production –  protein phosphorylation/dephosphorylation 2) Modelling of endocytic triggering and feedback loops 

Thank you Acknowledgements Dr. Andrew Goryachev CSBE, UoE Dr. Davide Marenduzzo School of Physics, UoE Prof. Sir Kenneth Murray The Darwin Trust of Edinburgh $ 

Experimental evidence Golebiewska et al., Mol Biol Cell 19:1663-1669, 2008 Golebiewska et al., Biophys J 91:588-599, 2006 FCS Fluorescence quenching experiments