membrane reconstitution systems & methods to model...

22.01.2019

1

Membrane reconstitution systems &

Methods to model membrane function ___________________________________

Milos Galic, PhD

Institute of Medical Physics & Biophysics

Dedicated Lecture Series - CRC1348 2019-01-22

slides: [email protected]

1

Date Type Topic Speaker

06.11.18 CRC Lecture 1. Biophysical properties of lipid bilayers: from polar lipids to lipid rafts Heuer

20.11.18 CRC Lecture 2. Composition and structure of biological membranes, interaction of proteins and sugars with membranes Krahn

04.12.18 CRC Lecture 3. Models of the plasma membrane – from the fluid mosaic to the picket fence model Schelhaas

18.12.18 CRC Lecture 4. Membrane biosynthesis & membrane homeostasis Gerke

08.01.19 CRC Lecture 5. Membrane isolation methods & methods for the analysis of biomembrane properties

Wedlich-Söldner

22.01.19 CRC Lecture 6. Membrane reconstitution systems & methods to model membrane functions Galic

05.02.19 CRC Lecture 7. Structure, function and activation of transmembrane proteins I (G-protein coupled receptors and channel proteins) Klingauf

19.02.19 CRC Lecture 8. Structure, function and activation of transmembrane proteins II (Signaling from transmembrane receptors) Klämbt

Overview

2

Structure of this lecture: Part I: Membrane reconstruction systems - Definitions & Theory - Current systems - Applications - Takehome message & Quiz Part II: Modeling membrane function - Definitions & Theory - Current systems - Applications - Takehome message & Quiz Part III: Literature & References

Overview

3

Part I / Definitions & Theory

Model membrane: ‚Membrane that is not derived from a cell‘ OR ‚Membrane that is ‘stolen‘ from a cell‘ ______________________________________________________ Membrane reconstituted system: ‚Proteins in model membranes‘

4

5

Water: • Dipol • Oxygen (χ= 3,5) partially negativ (δ−) • Hydrogen (χ = 2,2) partially positive (δ+)


… some energy considerations:

Hydrogenbridge (0.18nm)

Large contribution to entrophy of water!

6


22.01.2019

2

• Hydrophilic Object

• Commonly salt or polar solutions

• Soluble in water

• Hydrophobic Object

• nonpolare objects (e.g. long Alkyl-chains or aromatic rings)

• No electrostatic interactions with water

• Destruction of Hydrogen-bridges!!

7


• Amphiphilic molecule: unify hydrophobe and hydrophile fractions

• Spontaneus reaction (= free enthalpy is negative?!)

ΔG = ΔH - TΔS G = free Enthalpy H = Enthalpy (=U+pV) T = Temp S = Entropy

8


9

Part I / Definitions & Theory Part I / Current Systems

Types of ‚synthetic‘ model membranes • Vesicles (1961) • Planar bilayers (1962) • Solid supported bilayers (1986)

10 https://pubs.rsc.org/en/content/articlehtml/2009/sm/b901866b

https://pubs.rsc.org/en/content/articlehtml/2015/cp/c5cp00480b

SUV: 15-30nm LUV: 100-200nm GUV: >1µm

Part I / Current Systems


11 https://pubs.rsc.org/en/content/articlehtml/2009/sm/b901866b

https://pubs.rsc.org/en/content/articlehtml/2015/cp/c5cp00480b

SUV: 15-30nm LUV: 100-200nm GUV: >1µm



12 https://www.pnas.org/content/102/9/3272

http://www.mpikg.mpg.de/rl/P/archive3/veat03.pdf

Cholesterol DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

22.01.2019

3


13


https://pubs.rsc.org/en/content/articlehtml/2012/lc/c2lc20991h


14



15


How to reconstitute proteins into it?? • Detergent-based

• Vesicles • Nanodisc

https://www.sciencedirect.com/science/article/pii/S0076687903720047 16



• Vesicle • Nanodisc

https://link.springer.com/article/10.1007%2Fs00232-014-9666-8 17



• Vesicle • Nanodisc (2002, Sligar lab)

https://www.labome.com/method/Nanodiscs-Membrane-Protein-Research-in-Near-Native-Conditions.html https://pubs.acs.org/doi/abs/10.1021/nl025623k

MSP=Membrane Scaffold Protein (Derived from Apolipoprotein A1)

18


22.01.2019

4


• Vesicle • Nanodisc

• Alternatives

• Vesicles

https://www.nature.com/articles/s41598-018-33208-1#ref-CR4

amphipathic styrene maleic acid (SMA) co-polymer

19


BUT: No asymmetry in lipid composition Not physiological composition


Types of ‚borrowed‘ model membranes • Patch • Tether • GPMV (Giant Plasma Membrane Vesicles)

20


https://en.wikipedia.org/wiki/Patch_clamp https://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/#fancybox-4727-1

BUT: No asymmetry in lipid composition Not physiological composition



21


https://en.wikipedia.org/wiki/Patch_clamp https://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/#fancybox-4727-1



22


http://jcb.rupress.org/content/148/1/127



23


https://www.nature.com/articles/s41598-017-15103-3

What used for? • Membrane

• Structure • Organization • Microdomains

• Transmembrane channels

• Properties • Formation

• Protein IA with membrane

• Binding to membrane • Folding

• Protein-protein IA in membranes

• Clustering

24

Part I / Applications

22.01.2019

5








• Clustering

Baumgart, T., Hess, S., and Webb, W. " Nature. 425, 821-824 (2003)

Lo (perylene ) Ld (rho-DPPE)

25


https://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/








• Clustering

26


https://www.nature.com/articles/s41598-018-26606-y/figures/4 https://gucklab.com/wp-content/uploads/2016/05/for-methods-section-website-guck-lab1000.png








• Clustering

27


AFM What used for? • Membrane







• Clustering

https://chem.uiowa.edu/sites/chem.uiowa.edu/files/people/shaw/140402%20-%20JSG%20-%20QCM.pdf

Sensitivity ~ 2 ng/cm2

~ 12*1011 kDa/cm2

28


QCM








• Clustering

29


https://www.frontiersin.org/articles/10.3389/fmolb.2018.00074/full








• Clustering

30


http://science.sciencemag.org/content/361/6405/876 https://www.sciencedirect.com/science/article/pii/S0076687917301507

22.01.2019

6








• Clustering

31


http://science.sciencemag.org/content/361/6405/876 https://www.sciencedirect.com/science/article/pii/S0076687917301507

Part I / Takehome message & Quiz

Advantages of model membranes: Control of

Molecular components Solutions on both sites Size, shape & symmetry

Isolated biological process Homogenuous Reproducible

Cheap Stable Easy accesible for quantification

32

Disadvantages of model membranes: No membrane potential Nonphysiological lipid composition No assymetric leaflet composition

https://www.slideshare.net/stsmmentele/the-olympians-powerpoint1

Part I / Takehome message & Quiz

Quiz: What is this:

33


Overview

34

Variable Parameters • Bond Stretching • Angle Bending • Dihedral & Imporper Torsion • Van der Waals Interactions • Coulumbic interactions

Part II / Definition &Theory

35

ForceField

Molecular Dynamics From the molecular positions, the forces acting on each molecule are calculated; these are used to advance the positions and velocities through a small timestep, and then the procedure is repeated. Principal features: • Solution of Newton's equations of motion by a step-by-step algorithm. • Simulation times from picoseconds to nanoseconds. • The method provides thermodynamic, structural and dynamic properties.

Monte Carlo At each stage, a random move of a molecule is attempted; random numbers are used to decide whether or not to accept the move, and the decision depends on how favourable the energy change would be. Then the procedure is repeated. Principal features: • Sampling configurations from a statistical ensemble by a random walk algorithm. • No true analogue of time. • Possible to devise special sampling methods. • Provides thermodynamic and structural properties. Both methods employ system sizes from a few hundred to a few million molecules.

https://warwick.ac.uk/fac/sci/physics/research/theory/research/simulation/how/mdmc/ 36

Part II / Definition &Theory

22.01.2019

7

37

Part II / Current Systems

https://www.youtube.com/watch?v=AtDOJnVNC18 38


https://www.mdpi.com/2073-4360/5/3/890/htm 39


speedup of >500-fold

Duration: nanoseconds

Duration: microseconds

40

Part II / Applications

https://elifesciences.org/articles/37262 https://www.cell.com/biophysj/fulltext/S0006-3495(12)00389-X

https://aip.scitation.org/doi/pdf/10.1063/1.2372761

Pulling of a membrane tether (Martini Force Field):

41


Change in tension in membrane tether (ESPRESSO Force Field):

https://elifesciences.org/articles/37262 https://www.cell.com/biophysj/fulltext/S0006-3495(12)00389-X

https://aip.scitation.org/doi/pdf/10.1063/1.2372761 42

http://www.mpikg.mpg.de/rl/P/archive3/veat03.pdf https://www.pnas.org/content/105/45/17367

Phase separation (Martini Force field):


22.01.2019

8

43 https://www.nature.com/articles/s41586-018-0556-6#MOESM3

Opening of Holotoxin (GROMAC Force Field):

Part II / Applications Part II / Takehome message & Quiz

44 https://slideplayer.com/slide/7072473/

Advantages of MD studies: Have evolved into a mature technique to study molecular interactions

Limitations of MD studies: as good as the used force field (i.e. ability to correctly reproduce free energies)

Part II / Takehome message & Quiz

Quiz: What is the difference:

45

vs.

https://slideplayer.com/slide/7072473/


Overview

46

Literature & References

Part 1 • http://sites.psu.edu/cremer/wp-content/uploads/sites/3050/2013/04/65-Ed.pdf • https://people.chem.umass.edu/lkt/Courses/chem697a/LectureNotes/MattHolden1Feb.pdf • https://www.sciencedirect.com/science/article/pii/S0076687903720047 • https://link.springer.com/article/10.1007%2Fs00232-014-9666-8 • https://www.labome.com/method/Nanodiscs-Membrane-Protein-Research-in-Near-Native-Conditions.html • https://www.nature.com/articles/s41598-018-33208-1#ref-CR4 • Baumgart, T., Hess, S., and Webb, W. " Nature. 425, 821-824 (2003) • https://pubs.rsc.org/en/content/articlehtml/2012/lc/c2lc20991h • http://jcb.rupress.org/content/148/1/127 • https://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/ • https://www.pnas.org/content/102/9/3272 • http://www.mpikg.mpg.de/rl/P/archive3/veat03.pdf • https://www.nature.com/articles/s41598-018-26606-y/figures/4 • https://gucklab.com/wp-content/uploads/2016/05/for-methods-section-website-guck-lab1000.png • https://chem.uiowa.edu/sites/chem.uiowa.edu/files/people/shaw/140402%20-%20JSG%20-%20QCM.pdf • https://pubs.acs.org/doi/pdf/10.1021/acsomega.6b00395 • https://www.nature.com/articles/s41598-017-15103-3 • https://pubs.rsc.org/en/content/articlehtml/2009/sm/b901866b • https://pubs.rsc.org/en/content/articlehtml/2015/cp/c5cp00480b • https://en.wikipedia.org/wiki/Patch_clamp • https://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/#fancybox-4727-1 • https://www.slideshare.net/stsmmentele/the-olympians-powerpoint1 ___________________________________________________________________________________________________ Part 2 • https://warwick.ac.uk/fac/sci/physics/research/theory/research/simulation/how/mdmc/ • https://www.youtube.com/watch?v=AtDOJnVNC18 • https://www.mdpi.com/2073-4360/5/3/890/htm • https://elifesciences.org/articles/37262 • https://www.cell.com/biophysj/fulltext/S0006-3495(12)00389-X • https://aip.scitation.org/doi/pdf/10.1063/1.2372761 • http://www.mpikg.mpg.de/rl/P/archive3/veat03.pdf • https://www.pnas.org/content/105/45/17367 • https://slideplayer.com/slide/7072473/

47

membrane reconstitution systems & methods to model...

Documents