Journey of bacteriophage M13 major coat protein David Stopar University of Ljubljana Slovenia Interactions in macromolecular assemblies

Replication cycle of the bacteriophage M13 Structure of the major coat protein Topology of the major coat protein in the lipid bilayer Anchoring of the major coat protein in the lipid bilayer Journey of bacteriophage M13 major coat protein

Schematic representation of bacteriophage M13 replication cycle M13 E.coli F-pilus DNA gp 5 gp 8

Schematic structure of bacteriophage M13 filament

Model of the of bacteriophage M13 protein coat

Nearest-neighbour interactions between protein subunits in the phage particle k=11 k=6 k=0

Replication cycle of the bacteriophage M13 Structure of the major coat protein Topology of the major coat protein in the lipid bilayer Anchoring of the major coat protein in the lipid bilayer Journey of bacteriophage M13 major coat protein

Major coat protein structural domains in lipid bilayer 1-5 N-terminus 6-16 amphiphatic helix loop transmembrane helix C-terminus 1 50 Amino acid residue number

X-ray and NMR limitations for membrane proteins X-ray: “no” crystals of membrane proteins NMR: membranes are anisotropic systems high-resolution NMR limited to micellar systems solid-state NMR is needed for bilayer structure 13 C and 15 N-enrichment is needed (difficult and very expensive!)

2D NMR & DG analysis Papavoine et al. (1998) J. Mol. Biol. 282, M13 coat protein in SDS micelles ? ? ? ? Structure not possible in a membrane

Molecular Dynamics (MD) Approach Starting conformation for protein in POPC membrane from SDS (Papavoine et al., 1998).

Snap Shots of Protein Backbone 0 ns 0.5 ns 1 ns 1.5 ns 41 H- bonds 42 H- bonds 43 H- bonds The effects seen in MD are in the ESR time scale (ns)

Note: one label is measured at a time membrane-water interface M13 Coat Protein with Labels Attached

ESR Spin Label Information Position with structural restriction Position with no structural restriction N N O O O ESR spin label: 5-maleimido- proxyl

Sensitivity of spin labels for different protein sites 1 50 Amino acid residue number 2A zz Outer Splitting

Sensitivity of spin labels for different lipids PC22 PC14

Replication cycle of the bacteriophage M13 Structure of the major coat protein Topology of the major coat protein in the lipid bilayer Anchoring of the major coat protein in the lipid bilayer Journey of bacteriophage M13 major coat protein

Spin label relaxation times in DOPC bilayers A49C V31C G38C 1 50 Amino acid residue number

Relative quenching efficiency by oxygen and Ni 2+ Ni Amino acid residue number oxygen Ni 2+

Dependence of spin labeled DOPC acyl chains on the relaxation enhancement by oxygen and Ni 2+ Ni 2+ oxygen 1 50 Amino acid residue number

Location of the protein in the lipid bilayer as determined by fluorescence labeling 1 50 Amino acid residue number

Trp 26 Leu 14 NH 3 + Phe 45 Interface Hydrocarbon core 20 Å Lys 40 Topology of the major coat protein in the lipid bilayer

Replication cycle of the bacteriophage M13 Structure of the major coat protein Topology of the major coat protein in the lipid bilayer Anchoring of the major coat protein in the lipid bilayer Journey of bacteriophage M13 major coat protein

AEDANS max of the A7C protein mutants in lipid bilayers

AEDANS max in DOPC lipid bilayers 1 50 Amino acid residue number

Tryptophane max in DOPC lipid bilayers 1 50 Amino acid residue number

Bacteriophage M13 major coat protein anchors in the lipid bilayer C-terminus N-terminus Leu 14 Phe 11 Lys 8 Trp 26 Lys 40 Lys 43 Lys 44 Phe 42 Phe 45

Schematic representation of bacteriophage M13 replication cycle M13 E.coli F-pilus DNA gp 5 gp 8

Model of the major coat protein disassembly and assembly (1) (3) (2) N-terminus C-terminus

Acknowledgements Wageningen Marcus Hemminga Rob Koehorst Ruud Spruijt Werner Vos Cor Wolfs Göttingen Derek Marsh Kity A. Jansen Szeged Tibor Páli Ljubljana Ivan Mahne Janez Štrancar Milan Schara