Thermodynamic approaches to membranes and membrane interactions
This presentation is the property of its rightful owner.
Sponsored Links
1 / 46

Thermodynamic approaches to membranes and membrane interactions PowerPoint PPT Presentation


  • 45 Views
  • Uploaded on
  • Presentation posted in: General

Thermodynamic approaches to membranes and membrane interactions. Peter Westh NSM, Research Unit for Biomolecules Roskilde University [email protected] Thermodynamic approaches to membranes and membrane interactions. thermodynamics ?. Thermodynamics.

Download Presentation

Thermodynamic approaches to membranes and membrane interactions

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Thermodynamic approaches to membranes and membrane interactions

Thermodynamic approaches to membranes and membrane interactions

Peter Westh

NSM, Research Unit for Biomolecules

Roskilde University

[email protected]


Thermodynamic approaches to membranes and membrane interactions

Thermodynamic approaches to membranes and membrane interactions

thermodynamics ?


Thermodynamics

Thermodynamics

The science that deals with the relationship of heat and mechanical energy and the conversion of one into the other

Webster’s New Universal Dictionary 1979

A branch of physics that studies …… systems at the macroscopic scale by analyzing the collective motion of their particles using statistics

Wikipedia Jan. 2008

A macroscopic phenomenological discipline concerned with a description of the gross properties of systems

Kirkwood & Oppenheim: Chemical Thermodynamics, 1961

Macroscopic – gross properties – heat and mechanical energy – statistics - phenomenological

Relevance to molecular biology and biochemistry ?


Thermodynamics and bio molecules

Thermodynamics and (bio)molecules

  • Department of molecular thermodynamics…..

  • Hydrogen bond thermodynamics. Calculation of local and molecular physicochemical descriptors ”HYBOT-PLUS”

  • Thermodynamics of protein folding (Cooper 1999)

  • Thermodynamics of membrane receptors and channels (MB Jacson 1993)

How is that possible for an approach which is: ”phenomenological” “macroscopic” and describes “gross properties” ?

Thermodynamics is your x-ray glasses which enables you to screen the models and mechanisms which are suggested to rationalize the exploding amount of empirical biochemical knowledge (functional and structural)


Thermodynamics1

Thermodynamics

Is a wonderful structure with no contents

Aharon Katchalsky

For the (experimentally convenient) (P,T,ni) variable system

Koga (2007) Solution Thermodynamics: a differential approach. Elsevier.

For membranous (colloidal) systems perhaps a fourth variable: Area (dG/dA=g)


Thermodynamic studies of membranes a practical approach

Thermodynamic studies of membranes – a practical approach

  • Free energy of interaction

  • Calorimetry (energy of interaction):

    -scanning

    -titration

    -pressure perturbation

    -temperature modulated

  • Volumetric properties


Measuring free energy chemical potential changes of interactions

Measuring free energy (chemical potential) changes of interactions

Two experimental approaches:

  • Direct (model free)

    Measures the equilibrium distribution. For example dialysis equilibrium, freezing point depression, membrane osmometry, liquid-liquid partitioning, vapor pressure (ion selective electrode)

  • Indirect (model based, DG°)

    Any technique (e.g. spectral, hydrodynamic, thermal) which quantifies the concentration of a species in a proposed reaction. For example protein folding

    UN ,K=[N]/[U] and DG=-RTlnK

    Or membrane partitioning

    Peptide (aq)  peptide (membrane)

Andersen et al (2005) J Biochem Biophys Methd50, 269.


Free energy of interaction an example

Free energy of interactionan example

Water-phospholipid interactions (membrane hydration)


Direct measurements of the water vapor pressure

Direct measurements of the water vapor pressure

30.0

23.5

18.4

Adsorption isotherm POPC 25C

14.5

Temperature scanning, DMPC-water. Pressure difference between moist lipid and pure water.

Andersen et al (2005) J Biochem Biophys Methd50, 269.


Faster methods dynamic vapor sorption dvs

Faster methodsDynamic Vapor Sorption (DVS)


Sorption calorimetry

Sorption calorimetry

Heat (enthalpy) of adsorption is measured directly – the amount adsorbed is calculated from the evaporation enthalpy

Bagger et al (2006) Eu. Biophys. J.35, 367.


Sorption calorimetry1

Sorption calorimetry

DLPC 25C DMPC 27 C

Sorption isotherm

(net water affinity)

Heat of sorption

(DHw)

Markova et al. (2000) J Phys Chem B104, 8052


Lyotropic phase transitions

Lyotropic phase transitions

DLPC

DMPC

Markova et al. (2000) J Phys Chem B104, 8052


Calorimetry

Calorimetry

  • We measure the temperature dependence of the free energy (Gibbs Helmholtz eq.)

  • Most often, this is not explicitly used – we quantify the course of a process through the heat it produces


Membrane calorimetry

Membrane calorimetry

  • One of the oldest analytical principles still in use – Lavoisier had rather precise calorimeters by 1780.

  • Readily measured thermodynamic function.

  • Heat cannot be measured – temperature can.

  • Heat is NOT at state function – enthalpy and internal energy are.


Modern instruments itc 200

Modern instruments (ITC200)

No water bath

Noise level ~0.002mCal/sec or about 10nW.

The heat capacity is about 3 J/K – detection level ~0.1mJ

Hence the the thermal noise is about 1x10-7/3~3x10-8K !


Two types of calorimeters have revolutionized biochemical applications

Two types of calorimeters have revolutionized biochemical applications

  • Differential Scanning Calorimetry (DSC)

  • Isothermal Titration Calorimetry (ITC)


Classic use of dsc phase diagrams

Classic use of DSC phase diagrams

Blume (1983) Biochem. 22; 5436.

Schrader et al (2002) J.Phys.Chem. 106, 6581

Böckman et al (2003) Biophys J.85, 1647


Dsc and the lever rule

DSC and the lever rule

Binary membrane (two PCs) Phase diagram

Schrader et al (2002) J.Phys.Chem. 106, 6581

The ratio nF/nG quantifies the conversion of gel to fluid phase and is hence reflected in the callorimetric heat flow


Phase diagram for dope at low temperature and water content

Phase diagram for DOPE at low temperature and water content

Derived – and remarkably complex – phase diagram

Increasing water content

DSC data

Sharlev & Steponkus (1999) BBA1419, 229.


Mixed membrane systems phase behavior of phospholipid cholesterol systems

Mixed membrane systemsPhase behavior of phospholipid-cholesterol systems

19

25

30

Temperature

McMullen et al (1993) Biochem32, 516.

DMPC/POPC + 28 % Cholesterol

Luis Bagatolli http://scienceinyoureyes.memphys.sdu.dk


Alcohols depress the main p b l a phase transition temperature

Alcohols depress the main (Pb – La) phase transition temperature

Pressure Increases Tm – Le chateliers principle!


Alcohol and interdigitated phases

Alcohol and interdigitated phases

Rowe & Cutera (1990) Biochem. 29, 10398


Other compounds increase the main transition temperature

Other compounds increase the main transition temperature

Complex solute effects in Phosphatidyl enthanoamine

KSCN

Sucrose

Koynova, et al. (1997) Europ. Biophys. J. 25, 261


Binding and partitioning itc

DH  0

+

Binding and PartitioningITC

”Foreign molecules” bind or partitioning into membranes

We already saw the DSC approach to this – change in phase behavior reflects partitioning !

ITC approach – directly measure interaction:

Basic idea!


Technical overview power compensated itc after 1990

Technical overviewPower compensated ITC (after ~1990)

The feed-back system sustains a constant and very small DT between cell and reference.

Net refcell heat flow

Exothermic process is compensated out by (fast) adjustment of the feed-back heaters.

Electrical heater

  • +++

  • Fast responce, high sensitivity

  • - - -

  • Narrow applicability,

Feed-Back Control


Simple approach

Simple approach

Ligand in cell – titrate with membrane (NB the other way around won’t work since there is no saturation – it is partitioning between two phases)

Lipid membrane; 47.4mM

Octanol 0.61mM 1-octanol

OcOH depletion

Rowe et al (1998) Biochem. 37, 2430


Itc and partitioning data analysis

DH  0

+

ITC and partitioning:data analysis

Partitioning scheme: A(aq) ↔ A(mem)

Law of mass action: Kp=[Amem]/[Aaq]

Mass conservation:[A]tot=[Amem]+[Aaq]

Rowe et al. (1998) Biochem. 37; 2430.


Weaker interaction requires more complex procedures

Weaker interaction requires more complex procedures

Excess enthalpy, HE, of DMPC in 1-propanol

HE is the enthalpic contribution of DMPC towards the total enthalpy of the system

Hence, the slope HE/Calcohol is a measure of the enthalpy of DMPC-alcohol interactions

Note that HE vs Calcohol is not linear.

Trandum et al (1999) J.Phys.Chem.B103; 4751


Interaction of ethanol and dmpc dependence of phase and cholesterol

Interaction of ethanol and DMPCDependence of phase and cholesterol

Phase behavior

Interaction enthalpy

And partitioning coefficient

DMPC+10% Ganglioside Kp=87

DMPC+10% Sphingomyelin Kp=85

DMPC, Kp=28

DMPC+30% Cholesterol Kp=12

Partitioning of small alcohols scales with the membrane surface density

DeYoung & Dill (1988) Biochem. 27, 5281.

Cholesterol content

Trandum et al (1999) BBA, 1420, 179

Trandum et al (1999) BBA1420; 179

Trandum et al (2000) Biophys J78; 2486


Heat and thus calorimetry is the universal detector specialized methods show great versatility

Heat (and thus calorimetry) is the universal detector.Specialized methods show great versatility

A ”release protocol” for the determination of membrane permeation rates

10mM POPC vesicles injected into 150mM C10EO7 (upper) and 1mM C10EO7+10mM POPC (lower)

Heerklotz & Selig (2000) Biophys. J.81, 184.


Thermodynamic approaches to membranes and membrane interactions

Another asset of calorimetry is high resolutionMicelle formation and protein surfactant interactions

De-micellization of SDS

CMC readily determined to within 10-50mM

Otzen et al In press


Thermodynamic approaches to membranes and membrane interactions

Another asset of calorimetry is high resolutionMicelle formation and protein surfactant interactions

Andersen et al Langmuir in press


A new generation of dsc temperature modulated dsc

A new generation of DSCTemperature Modulated DSC


A linear gradient in t with a sine wave or zigzag superimposed

A linear gradient in T with a sine wave or zigzag superimposed

Temperature

Heat Flow


In phase and out of phase heat capacity single out different response relaxation processes

In-phase and out-of-phase heat capacity single out different response/relaxation processes


Pressure perturbation dsc

Pressure perturbation DSC

Measures

HEAT OF COMPRESSION

Which is tantamount to

THERMAL EXPANSIVITY


Ppc two examples from biophysics

PPC – two examples from biophysics

Melting of egg sphingomyelin. Conventional DSC and PPC. DH=30.5 kJ/mol, DV=21 ml/mol

Thermal denaturation of two globular proteins

Area equals the volume change, DV, for the denaturation

Heerklotz (2004) J. Phys Condens Matter16, R441


Volumetric properties

Volumetric properties

  • V=dG/dp

  • Readily measured by vibrating tube densitometry.

  • ”Structural interpretation” and relationship to physical dimensions


Vibrating tube densitometry

Vibrating tube densitometry

Hollow quartz U-tube.

Volume 1 ml

Thermostatted 0.001 K

Hook’s law

Period measured to 1nsec

Calibrate against air and water

F~300Hz

For liqiuds (and gasses):

Specific volume (density) measured to within 10-6 to 10-5 cm3g-1 (g cm-3)


Vibrating tube densitometry1

Vibrating tube densitometry


Volume density of pure membranes

Volume (density) of pure membranes

  • DMPC @ 30C V~0.978 cm3/g (d~1.022 g/cm3)

  • DV @ Tm 4%

  • Monounsaturated PC membranes (e.g. both cis and trans DOPC) have higher volumes (~1.020 to 1.050 cm3/g @ 30C.

  • Polyunsaturated PC (like di-linolenoyl PC i.e. 18:3/18:3-cis-D9,12,15) have volumes similar to saturated PC

Volume (density) of mixtures

  • Illustrates how the different species pack

  • May benchmark MD simulations

Nagle & Wilkinson (1978) Biophys J 23, 159

Trandum & Westh (2000) J Phys Chem B 104, 11334


Molecular packing experiment vs simulation

Molecular packing:Experiment vs. simulation

Voronoi assignments of molecular volumes

DVhexanol (exp)= 4.2 ml/mol

DVhexanol (exp)= 3.9 ml/mol


Densitometry on membrane of membrane solute systems

Densitometry on membrane of membrane-solute systems

A typical sample consists of

97% water

2.9% Phospholipid

0.1% fatty acid

Measured specific volume V


Molecular packing of alcohols in dmpc

Molecular packing of alcohols in DMPC

DV=Vapp-V

(standard pure alcohol)

Volume of each component

Lipid

alcohol

water

Aagaard et al 2005


Closing

Closing

Although thermodynamic functions reflects ”macroscopic properties” they effectly elucidate molecular aspects of membranes and membrane interactions.

Calorimetry is the most precise and versatile experimental approach.


  • Login