Lecture 2 What do we do? (projects in the Sukharev Lab)

1. Lecture 2 What do we do? (projects in the Sukharev Lab) Reading for the next classes: Chapter 2 (Chemical foundations)

2. What is the wavelength if the frequency of atomic oscillations f = 1014 s-1 c = f ∙l c = 3 ∙108 m/s (in vacuum) l = c/ f = 3 ∙ 10-6 m = 3 mm infrared 470 nm = blue 530 nm = green 600 nm = yellow 650 nm = orange 700 nm = red >800 nm = infrared

3. wavenumber = 1/l The stiffer is the bond the higher is frequency and smaller wavelength It also depends on the mass of the atom

4. Basic Senses • Vision • Taste • Smell • Hearing, Equilibrium and Touch • Temperature sensation

5. Mechanical forces in the body Force detection Faint sound~10-4 N/m2 Systolic pressure ~104 N/m2 Postural pressure on an intervertebral disk ~105 N/m2 Osmotic pressure (0.1M sugar gradient) = 2.4x105 N/m2 - It can’t be one receptor!!! Force generation Molecular motors? Yes, but what exactly drives the tissue boundary formation and organogenesis in development: how do the feedback loops work?

6. These are cartoons of the gating process. There is no structural information about any of the eukaryotic channels. However, such information is available for two prokaryotic channels, MscL and MscS

7. ?

8. γ H2O γ πOSM H2O Bacterial osmoregulation Open MS channels (γ = tension) Osmotically balanced medium Low osmolarity medium Preventlysis AfterBritten & McClure, 1962

9. MscS MscL MscK Patch-clamp recording of channels with a glass pipette

10. Tb MscL (Chang et al. 1998) Eco MscS (Bass et al., 2002)

11. The gating mechanism of MscL (E. coli model) H.R Guy

13. Lipids can be distorted near the edge of the flattened protein (due to the thickness mismatch), but their elastic recoil may help closing the channel.

14. Two-State Model A Boltzmann equation for the ratio of open and closed state probabilities, it dictates the dose-response relationship, i.e. fraction of open channels versus tension (gamma).

15. Modeled expansion of MscL well corresponds to experimental data 18 nm2 ~41 nm2 DAmodel = 23 nm2 DAexp = 20 nm2 Pore diameter predicted from conductance ~ 2.9 nm

16. I32C-N81C I24C-G26C F10C-F10C F7C-F7C L121C-L122C L128C-L129C

17. I32C-N81C A20C-L36C I3C-I96C L121C-L122C L128C-L129C

18. The Crystal Structure of MscS (286 aa) from Bass et al., Science, 298(2002)1582

19. The kink region in MscS (electron densities) Bass et al, 2002

20. MscS-like channels are found in most organisms with walled cells

21. Mutations in the Arabidopsis msl2 and msl3 genes lead to swelling and improper division of plastids From Haswell and Meyerowitz, 2006

22. Cross-section of the transmembrane domain and gate regions of MscS

23. MscS constriction is largely dehydrated based on Molecular Dynamics simulations

24. Gating by ‘bubble’ implies capillary evaporation in the hydrophobic confinement

25. Hydrophilic substitutions favor pore wetting in simulations and strongly influence the speed of transitions in experiments

27. Energies and expansion areas from 4-state analysis C3/O* WT (2-state) DE 23.4 kT 24 kT DA 22.8 nm2 17.7 nm2 O4 C2 A98S DE 12.1 kT 14.0 kT DA 13.7 nm2 13.5 nm2 C1

28. Key stages in model development

29. Transitions between the functional states reveal distinct conformations of the pore lining TM3 helices Alternate Kink at G121 Kink at G113

30. WT G113A G121A G113A/G121A Double alanine mutant traps the open state • G113A/G121A • High helical propensity at both G113 and G121 kinetically traps MscS in the open state Straight TM3 helices are a feature of the open state

31. Separation of peripheral helices

33. F68S mutant is prone to fast and silent inactivation