Mechanical Properties of Tubulin Monomers Simulated via Molecular Dynamics and AFM Experiments

1. Tubulin Monomer Mechanical Properties Obtained by Simulating Atomic Force Microscopy Experiments Using Molecular Dynamics Supervisors: Prof. A. Redaelli Ing. S. Monica PhD student: Søren Enemark

2. 30 nm α-Tubulin β-Tubulin 18 nm Introduction: What am I talking about? αβ-tubulin dimer Hetero dimer 2 x ~450 residues Subunit in MTs Microtubules (MTs) Length ~ 1-10μm Cylinder-shaped Lattice structure Rot. + trans. symmetry

3. Introduction: What are microtubules good for? • MTs’ main functions: • Structural elements • Intracellular transport • Cell division

4. Lateral Longitudinal Modeling: Stress-strain directions Single monomer mechanical properties MT mechanical properties Compression Elongation 4 tests for each monomer

5. Modeling: Stress-strain directions (cont’d) Directions depend on the MT structure: Longitudinal interactions Along straight line 13.8° Lateral interactions Atomic structure by K. Downing Basic structure 1JFF.pdb Fitted to MT structure data Monomer centre-of-mass (CM) End view on MT Along lines towards CM of lateral monomers

6. EM Steepest descent (1000 steps) 3. Procedure: Preparing the structure 1. Monomer structure extracted Arrange in box w/ SPC water 2. Dodecahedron box System size ~ 37,000 (SOL) + 4,500 (monomer) MD Pull groups position restraints 4. Parameters Twin-range cut-off rvdw = 1.4 rcoulomb=1.4 rlist= 0.8 nstlist = 5 Berendsen thermostat SOL monomer Two groups: tau= 0.1 ps Tref = 300 K All-bonds constraints, Lincs

7. Preparing AFM-like MD Pull groups: • residues < 8 Ǻ from interacting monomer • In longitudinally tests: 731 and 675 atoms • In laterally tests: 188 and 198 atoms Retain interface structure, but generate new configurations Position restraint pull groups 1000 ps 0 ps

8. S’ 0.3 x (nm) 0.2 P’ 0.1 0 600 300 200 500 400 100 t(ps) -0.1 -0.2 P -0.3 S AFM-like MD – how to measure the stiffness AFM-like Method Typical results (pulling) Pull group P1 Pull group P2 S1 P1 Spring S1 Spring S2 P2 S2 Spring stiffness 103 kJ/(nm2 mol) = 1.67 nN/nm Spring velocity (7-11 simulations) v=5 10-3 nm/ps

9. 1.25 1.25 1.00 1.00 0.75 0.75 a= 4.6 nN/nm b= 0.2 nN -0.1 -0.1 0 0 0.1 0.1 0.2 0.2 0.3 0.3 Single monomer - MD results -tubulin – pulling - longitudinally Linear fit y(x) = a x + b F (nN) 0.50 v=5 10-3 nm/ps 0.25 l (nm) F (nN) a= 5.2±0.4 nN/nm b= 0.4±0.1 nN v=5 10-3 nm/ps l (nm)

10. a 6.6± 0.3 a 3.7± 0.5 a 3.2± 1.0 a 3.8± 0.9 a 5.4± 1.3 a 5.8± 0.6 a 5.2± 0.4 a 6.4± 0.4 b 0.0± 0.1 b 0.4± 0.1 b -1.1± 0.5 b 0.9± 0.1 b 1.0± 0.3 b 1.6± 0.2 b -0.5± 0.3 b -0.3± 0.1 Single monomer - MD results Longitudinally Laterally v=5 10-3 nm/ps Elongation Compression Elongation Compression α-tubulin β-tubulin Monomers are more rigid longitudinally than laterally Monomer might be less rigid under elongation than compression α-tubulin might be less rigid then β-tubulin longitudinally, but more rigid laterally

11. k CM k k CM k CM 2k CM Funded Simplified MT model – a bed of springs α-, and β-tubulin stiffness & monomer-to-monomer interactions MD Elastic constants FEM Axial tests on 1 μm MT “Tubulin Monomer Interaction Study by Molecular Dynamics Simulation” POSTER: Marco Deriu et al. EST Marie Curie programmecontract No. MEST-CT-2004-504465 Active BIOMICS STREP projectcontract No. NMP4-CT-2004-516989