PHASE DIAGRAMS OF 3-COMPONENT, DOMAIN-FORMING MEMBRANE MODEL SYSTEMS by cometjunkie44

VIEWS: 0 PAGES: 25

									PHASE DIAGRAMS OF 3-COMPONENT, DOMAIN-FORMING
           MEMBRANE MODEL SYSTEMS




               Gerald W. Feigenson
               Field of Biophysics
               Cornell University
                     ORGANIZATION OF TALK

PART I: FOR ONE 3-COMPONENT MIXTURE, DPPC/DLPC/CHOLESTEROL,
PHASE DIAGRAM IS ~ COMPLETE


PART II: OTHER 3-COMPONENT MIXTURES HAVE DIFFERENT PHASE
BEHAVIOR
  A. GEL LIPID: SPHINGOMYELIN BEHAVES DIFFERENTLY THAN DSPC

  B. FLUID LIPID: DOPC-CONTAINING MIXTURES SHOW “MACROSCOPIC” Lo + Lα;
  POPC- OR SOPC-CONTAINING MIXTURES, Lo + Lα NOT “MACROSCOPIC”
  C. PHASE COEXISTENCE IS NOT THE SAME AS NONIDEAL MIXING

  D. THE CHOLESTEROL DEPLETION EXPERIMENT, PHASE BEHAVIOR VIEWPOINT
4 BILAYER PHASES EXAMINED IN THIS STUDY




                 A RAFT?! DIFFERENT KINDS?! LIFETIME?
                 SIZE? COMP? PROTEINS KEPT IN/OUT?
        A GENERIC 3-COMPONENT PHASE DIAGRAM

ALL MIXTURES OF
COMPONENTS A, B, AND C    PHASES: a,   b, & c
AT CONSTANT
                          COULD BE NO 2-PHASE OR 3-PHASE
TEMPERATURE
                          COEXISTENCE REGIONS, OR SEVERAL
              METHODS WE USE TO DETECT PHASES
VESICLE IMAGING: CONFOCAL FLUORESCENCE MICROSCOPY
• SEPARATION OF COEXISTING PHASES, DISTANCE SCALE > ~ 300nm

X-RAY DIFFRACTION
• STRUCTURE

ELECTRON SPIN RESONANCE
• RATE OF MOLECULAR MOTION; ORDER

FLUORESCENCE RESONANCE ENERGY TRANSFER
• INTERMOLECULAR DISTANCE (SEPARATION > ~ 60 ANGSTROM)

FLUORESCENCE EXCIMER/MONOMER RATIO
• CRUDE MEASURE OF BILAYER ORDER

FLUORESCENCE CORRELATION SPECTROSCOPY
• RATE OF TRANSLATIONAL DIFFUSION

SIMPLE FLUORESCENCE
• DIFFERENT FLUORESCENCE Q.Y. IN DIFFERENT PHASES

DIFFERENTIAL SCANNING CALORIMETRY
• TRANSITION TEMPERATURE; ENTHALPY
LIPIDS
 LIPIDS
STUDIED
STUDIED
SOME PROBES
   OF LIPID
   BILAYER
    PHASE
  BEHAVIOR
A SOLVED PHASE DIAGRAM?!          16:0,16:0-PC/12:0,12:0-PC/CHOLESTEROL

                                                  DPPC/DLPC/CHOL
                             CHOLESTEROL
     2-PHASE, CRYSTAL + Lo




           1-PHASE, Lo
                                                3-PHASE, Lα + Lo + Lβ

  2-PHASE, Lα + Lo
                                                     2-PHASE, Lo + Lβ

1-PHASE, Lα                                              1-PHASE, Lβ

                                                             2-PHASE,
2-PHASE,                                                     Lβ + Lβ’
Lα + Lβ


MELTS    12:0,12:0-PC                              16:0,16:0-PC MELTS
~ -1oC                                                          42oC
LOW ANGLE X-RAY DATA
DPPC/CHOL MULTIBILAYER STACKS
                        16:0,16:0-PC/CHOLESTEROL WIDE-ANGLE X-RAY
                       SCATTERING: COEXISTING PHASES OF Lβ AND Lβ’
                       TILTED HYDROCARBON CHAINS IN A
                       DISTORTED HEXAGONAL LATTICE:
                       ONE SHARP LINE & ONE BROAD LINE


                              4.22-1
                                                      4.16-1
SCATTERING INTENSITY




                                                           CHOLESTEROL
                                                           MOLE FRACT.
                                                                 0.0
                                                                 0.018
                                                                 0.035
                                                                 0.044   TILTED CHAIN PHASE Lβ’
                                                                 0.052
                                                                         DISAPPEARS AS [CHOL]
                                                                 0.061   INCREASES
                                                                 0.070
                             4.4-1     o -14.2
                                                 -1      4.0-1
                                       A
MICROSCOPY IMAGES OF GIANT VESICLES: BOUNDARIES
USEFUL, BUT THIS AND PHASE IDENTIFICATION
IN NOT THE PHASE
DIAGRAM!
ESR SPECTRA AND SIMULATIONS: COEXISTING
  PHASES, AND PHASE CHARACTERIZATION
ACYL CHAIN ORDER PARAMETER, So
• [CHOL] = 0, So HAS 2 COMPONENTS: 2 PHASES
• [CHOL] = 0.2, So HAS 2 COMPONENTS: 2 PHASES
FLUORESCENCE RESONANCE ENERGY TRANSFER, FRET
FRET CAN REVEAL PHASE TRANSITIONS
di-PYRENE-PC CAN FORM EXCIMERS: MEASURE
     OF “LOCAL” [PYRENE] AND MOBILITY
di-PYRENE-PC EXCIMER/MONOMER RATIO:
 • ONE REGION FAVORING EXCIMER;
 • ONE REGION OF LOW EXCIMER (Lβ)
   HOW CAN THE PHASE DIAGRAM BE USED IN MODELS?
A. χCHOL = 0.66 (PUBLISHED)
HEADGROUPS COVER NONPOLAR PATCH UP TO
CHOL-CHOL-CHOL

B. χCHOL = 0.17 (0.16 - 0.175)
EACH CHOL SURROUNDED BY ~ ONE LAYER OF Lα PC

C. χCHOL = 0.20 (0.18 - 0.20)
EACH CHOL SURROUNDED BY ~ ONE LAYER OF Lβ PC
**BUT EACH Lβ PC CAN COVER MORE CHOL THAN CAN
  EACH Lα PC

D. χCHOL = 0.30 (0.27 - 0.30)
NO MODEL! χCHOL = ~ 0.30 IS MAXIMUM CHOL CONC IN Lo PHASE

E. χCHOL = 0.07
Lβ’ ACCEPTS ~ NO CHOLESTEROL OR DLPC. Lβ DOES ACCOMMODATE DLPC OR CHOL
                              C                  C
                                   C        C
C   = CHOLESTEROL
                                       C
    = PC, SLIGHTLY PERTURBED C                    C
    TO SHIELD CHOL                C        C
                                      CC
                              C                C
                                          C
                                     C
                                 C            C
           VERY DIFFERENT PHASE BEHAVIOR:
          18:0,18:0-PC/18:1,18:1-PC/CHOLESTEROL

DSPC/DOPC/CHOL                         THIS IS NOT A
                                       PHASE DIAGRAM!




   MACROSCOPIC Lo + Lα




                                             MACROSCOPIC
                                             Lβ + Lα
 DIFFERENT YET: SPHINGOMYELIN/18:1,18:1-PC/CHOLESTEROL
Sph/DOPC/CHOL
EXTENSIVE REGION OF Lo + Lα
MACROSCOPIC PHASE
SEPARATION




NO MACROSCOPIC
SEPARATION OF
Lβ + Lα !
  DO OTHER LIPID MIXTURES SHOW MACROSCOPIC PHASE
                SEPARATION OF Lo + Lα?
DSPC/POPC/CHOL




                                 HOWEVER: REPLACE 15%
NO MACROSCOPIC                   OF POPC BY DOPC:
PHASE SEPARATION                 MACROSCOPIC
                                 SEPARATION OF Lo + Lα!
                                 IN 3-COMPONENT
                                 MIXTURES CONTAINING
                                 CHOLESTEROL, ACYL
                                 CHAIN DETAILS OF THE
                                 “FLUID LIPID” ARE KEY
MACROSCOPIC
Lα + Lβ
SPH/SOPC/CHOL: NO MACROSCOPIC PHASE SEPARATION
UNTIL SOPC PARTIALLY REPLACED BY DOPC




                   1-PHASE? NO MACROSCOPIC
                   PHASE SEPARATION


                               REPLACE 30% OF SOPC WITH
                               DOPC MACROSCOPIC
                               SEPARATION OF Lo + Lα
CHOLESTEROL DEPLETION EXPERIMENT: WHAT REALLY HAPPENS?!




     ASSUME THIS
     STARTING
     COMPOSITION

    REMOVE ONLY
    CHOLESTEROL
                            SUMMARY
1. “COMPLETE” PHASE DIAGRAM DETERMINED FOR DPPC/DLPC/CHOL. FOR
THIS OR ANY 3-COMPONENT MIXTURE: SOLVING THE PHASE DIAGRAM
REQUIRES DIFFERENT METHODS AND MANY SAMPLE COMPOSITIONS.
2. IN DPPC/DLPC/CHOL COEXISTING PHASES OF Lo + Lα EXIST WITH
DIMENSIONS < ~ 300 nm IN A SMALL COMPOSITION SPACE OF THE MIXTURE,
AND PROBABLY IN OTHER BILAYER MIXTURES.
3. THE Lo PHASE, OR “RAFT PHASE”, IS MACROSCOPIC IN MIXTURES THAT
CONTAIN 18:1,18:1-PC (DOPC).
4. MACROSCOPIC SEPARATION OF Lo + Lα CAN BE INDUCED BY ADDING
18:1,18:1-PC: THE Lα PHASE LIPID CONTROLS DOMAIN SIZE?!

                        CURRENT WORK
1. TITRATE POPC/DOPC IN SPH/CHOL: TRANSITION FROM NANO TO
MACROSCOPIC GRADUAL? ABRUPT? MEASURE SIZE?!
2. MIXTURES THAT CONTAIN NATURAL (POLY)UNSATURATED ACYL CHAINS,
e.g. C22:6 OR C18:2 --- MACROSCOPIC OR NANOSCOPIC Lo + Lα ?
3. FOR 2-PHASE COEXISTENCE OF Lo + Lα --- CAN WE FIND THE
THERMODYNAMIC TIE LINES: WHICH COMPOSITIONS ACTUALLY COEXIST?
4. HOW DO MEMBRANE PROTEINS DISTRIBUTE BETWEEN COEXISTING
PHASES?
                                    RESEARCHERS
Feigenson group:
Fred Heberle (technician; FRET)
David Stringer (technician; FRET)         Jeff Buboltz (Colgate; FRET & X-ray)
Jing Wu
Jiang Zhao                                Juyang Huang (Texas Tech; model & X-ray)
Grace Huang
Geoff Hunt
                                          Paul Klawitter (SUNY Upstate Med. Cntr.)
Nieraj Jain
Chris Barry
Greg Costanza                             And thanks to Ben Widom!


Freed group:
Yun-Wei Chiang (grad student; ESR)


Webb group:
Tobias Baumgart (postdoc; GUV FCS)
Jonas Korlach (postdoc; GUV FCS)

$$$   NSF, ACS-PRF

								
To top