Lecture 2

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

Reading for the next classes: Chapter 2 (Chemical foundations)

frequency of atomic oscillations f = 1014 s-1

What is the wavelength if the

c = f ∙

= c/ f = 3 ∙ 10-6 m = 3 m

c = 3 ∙108 m/s (in vacuum)

infrared

470 nm = blue 530 nm = green600 nm = yellow 650 nm = orange700 nm = red >800 nm = infrared

wavenumber = 1/

The stiffer is the bond the higher is frequency and smaller wavelengthIt also depends on the mass of the atom

Basic Senses

• Vision

• Taste

• Smell

• Hearing, Equilibrium and Touch

• Temperature sensation

Mechanical forces in the body

Force detectionFaint 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 generationMolecular motors? Yes, but what exactly drives the tissue boundary formation and organogenesis in development: how do the feedback loops work?

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

?

Bacterial osmoregulation

Osmoticallybalanced medium

Low osmolaritymedium

Open MS channels

(γ = tension)

HH22OO

HH22OO

ππOSMOSM

γγ

γγ

Prevent lysis

After Britten & McClure, 1962

MscS MscL

MscK

Patch-clamp recording of channels with a glass pipette

Tb MscL (Chang et al. 1998)

Eco MscS (Bass et al., 2002)

The gating mechanism of MscL (E. coli model)

H.R Guy

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.

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).

Amodel = 23 nm2

18 nm2 ~41 nm2

Aexp = 20 nm2

Pore diameter predicted from conductance ~ 2.9 nm

Modeled expansion of MscL well corresponds to experimental data

F7C-F7C

F10C-F10C

I24C-G26C

I32C-N81C

L121C-L122C

L128C-L129C

L121C-L122C

L128C-L129C

I32C-N81CA20C-L36C

I3C-I96C

The Crystal Structure of MscS (286 aa)

from Bass et al., Science, 298(2002)1582

Bass et al, 2002

The kink region in MscS (electron densities)

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

From Haswell and Meyerowitz, 2006

Mutations in the Arabidopsis msl2 and msl3 genes lead to swelling and improper division of plastids

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

MscS constriction is largely dehydrated based on Molecular Dynamics simulations

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

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

C1

C2

C3/O*

O4

WT (2-state)E 23.4 kT 24 kTA 22.8 nm2 17.7 nm2

A98SE 12.1 kT 14.0 kTA 13.7 nm2 13.5 nm2

Energies and expansion areas from 4-state analysis

Key stages in model development

Transitions between the functional states reveal distinct conformations of the pore lining TM3 helices

Alternate Kink at G121Kink at G113

Double alanine mutant traps the open state

•G113A/G121A

•High helical propensity at both G113 and G121 kinetically traps MscS in the open state

WT

G121AG113A

G113A/G121A

Straight TM3 helices are a feature of the open

state

Separation of peripheral helices

F68S mutant is prone to fast and silent inactivation