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

2. c = f ∙l c = 3 ∙108 m/s (in vacuum) l = c/ f = 3 ∙ 10-6 m = 3 mm
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 nm = green
600 nm = yellow 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
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. Bacterial osmoregulation
Open MS channels
(γ = tension)
Osmotically
balanced medium
Low osmolarity
medium
Prevent lysis
H2O
πOSM γ
Mention Levina 1999.
Main finding 85% of the aa pool can be lost on osmotic downshock without compromising viability
After Britten & 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 kT kT
DA nm nm2 O4 C2
A98S
DE 12.1 kT kT
DA 13.7 nm nm2 C1

28. Key stages in model development

29. Transitions between the functional states reveal distinct conformations of the pore lining TM3 helices
Multiple attempts to visualize the transitions between states revealed interesting complexity in the TM3 pore lining helices
G113 kink disappeared exactly at 12nm2 and 16A pore.
Start with the crystal structure. Non-conducting. Find the open state.
Using extrapolated motion expanded the crystal structure.
Open state features XX Area and ~1.2 nS conductance from experiments.
Andiry noticed as well as other groups that G113 straightens.
From the open state use extrapolated motion to close.
60%, more than half of the time G121 kink formed. Some times both partially formed, but rarely did G113 reform.
Alternate Kink at G121
Kink at G113

30. Double alanine mutant traps the open stateWT
G121A
G113A
G113A/G121A
G113A/G121A
High helical propensity at both G113 and G121 kinetically traps MscS in the open state
Growth curves collected by Naili show toxic phenotypes.
Straight TM3 helices are a feature of the open state

31. Separation of peripheral helices
Inactivated state is a good starting point because of its smaller ‘footprint’ > explains why it stays open at very high pressures

33. F68S mutant is prone to fast and silent inactivation