Title: Facilitated Phospholipid FlipFlop Across Bilayer Membranes
1Facilitated Phospholipid Flip-Flop Across Bilayer
Membranes J. Middleton Boon Department of
Chemistry and Biochemistry University of Notre
Dame April 9, 2003
2Flip-Flop
Boon and Smith, Curr. Opin. Chem. Biol. 2002, 6,
749-756.
3SM
PC
PS
PE
Op den Kamp, Annu. Rev. Biochem. 1979, 48,
47-71. Boon and Smith, Med. Res. Rev. 2002, 22,
251-281.
4flippase
Zwaal and Schroit, Blood 1997, 89, 1121-1132.
5SM
PC
PS
PE
- I. PC Scramblases
- 1) TREN-Derived
- 2) Steroid-Derived
- II. PE Scramblases
- III. PS Scramblases
6SM
PC
PS
PE
I. PC Scramblases 1) TREN-Derived 2)
Steroid-Derived
71) TREN-Derived Scramblases
Boon and Smith, J. Am. Chem. Soc. 1999, 121,
11924-11925. Boon, et al., J. Org. Chem. 2001,
67, 2168-2174.
8Flip Assay
Add dithionite
Add detergent
POPC
NBD-X
POPC (25 µM)/NBD-X (0.5)/pH 7.4, 25 C
9NBD-PC Flip Across Vesicle Membranes
POPC 25 µM (0.5 NBD-PC)
Scramblase 38 µM
10POPC Binding
H2O
X
H2O
X
H2O
X
NH
CDCl3, 25 C
11Titration Isotherms
CDCl3, 25 C POPC
Sulfonamide (2100 80 M-1) gt Amide (30 4 M-1)
gt Ester (lt1 M-1)
12Crystal Structures
Sulfonamide
Amide
Dr. S. Brown
13pKa Measurements
991 CD3OD/D2O, 25 C
Sulfonamide 4.00 0.01 Amide 5.52 0.02
Ester 3.75 0.01
14Membrane Interactions
Rh emission
Dr. T. Lambert
NBD emission/ Rh excitation
NBD excitation
POPC 25 µM (0.6 Rh-PE)
NBD-sulfonamide 38 µM
15Add detergent
Add NBD-sulf
POPC 25 µM (0.6 Rh-PE)
NBD-sulfonamide 38 µM
16Membrane Partitioning
Dr. T. Lambert
? POPC (25 µM)/Dansyl-sulfonamide (5 mol )
17Translocation Mechanism
Sulfonamide associates more strongly with PC
head-group than amide 1) Increased acidity of
sulfonamide NHs 2) Molecular geometry that is
able to form tridentate complexes with phosphate
oxygen 3) Smaller fraction protonated at pH 7.4
18Erythrocyte Morphology
DLPC
Bilayer Couple Theory A membrane with proteins
and polar lipids distributed asymmetrically can
respond differently to perturbation while
remaining coupled to each other.
19 Control DLPC added
pH 7.4, 37 C
Boon and Smith, J. Am. Chem. Soc. 2001, 123,
6221-6226.
20Sulfonamide
Amide
Scramblase 35 µM 2 h, pH 7.4, 37 C
Ester
210
1
2
5
4
3
Morphological Index
0
-1
-2
-3
-4
Ferrell, et al., Biochemistry 1985, 24,
5406-5416. Daleke and Huestis,
Biochemistry 1985, 24, 5406-5416.
22Morphological Index Curves
Scramblase 35 µM
pH 7.4, 37 C
Average of 3 separate experiments
23NBD-PC Flip Across Erythrocyte Membranes
Lipid 2 mM (1 mol NBD-PC), Scramblase
40 µM pH 7.4, 37 C,
Average of 3 separate experiments
24Peroxisome Proliferator-Activated Receptor ?
(PPAR?)
AzPC must translocate across the cell membrane,
because the presence of the sulfonamide
scramblase increased the expression of
PPAR-responsive genes!
Davies, et al., J. Biol. Chem. 2001, 276,
16015-16023.
252) Second Generation Steroid-Derived Scramblases
Dr. T. Lambert
Lambert, Boon, et al., J. Am. Chem. Soc. 2002,
124, 5276-5277.
26NBD-PC Flip Across Vesicle Membranes
Half-lives
NBD-PC 18 min NBD-PE 50 min NBD-PS 160
min
POPC 25 µM (0.5 NBD-PC) 5 mol
scramblase Average of 3 separate
experiments
27UV Binding Study
991 CHCl3/CH3OH, 25 C POPC (1.2
0.1) x 105 M-1
28Methyl Ester Version
Dr. T. Lambert
29Erythrocyte Morphology Study
Scramblase 40 µM
pH 7.4, 37 C
Average of 3 separate experiments
30NBD-PC Flip Across Erythrocyte Membranes
Lipid 2 mM (1 mol NBD-PC) Scramblase 40
µM pH 7.4, 37 C
Average of 3 separate experiments
31II. PE Scramblases
SM
PC
PS
PE
Boon, et al., Chem. Commun. 2002, 260-261.
32NBD-Lipid Flip Across Vesicle Membranes
Dr. R. Shukla
POPC 25 µM (0.5 NBD-X)
Scramblase 100 µM
33NBD-Lipid Flip Across Vesicle Membranes
Dr. P. Scrimin
POPC 25 µM (0.5 NBD-X) Scramblase
8 µM Average of 3 separate
experiments
34III. PS Scramblases
SM
PC
PS
PE
Boon, et al., J. Am. Chem. Soc. 2003, submitted.
35NBD-PS Flip Across Vesicle Membranes
Dr. T. Lambert
Dr. A. Davis
POPC/Chol (73) 25 µM (0.5 NBD-PS)
Scramblase 1.25 µM
Average of 3 separate experiments
Dr. T. Lambert
36Half-Lives for NBD-Lipid Translocation (min)
PS
PC
PE
PG
PA
30 120 16 lt1 lt1
gt180 gtgt180 gtgt180 120 120
gtgt180 gtgt180 gtgt180 gt180 gtgt180
37Translocation Mechanism
38Binding of Annexin V-FITC
39Flow Cytometry - Erythrocytes
0.8
39
0.8
Number of Cells
FITC-Annexin V Fluorescence Intensity
10 µM scramblase 1.5 x 107 cells/mL
3 h, pH 7.4, 37 C
400.8
39
0.8
Number of Cells
FITC-Annexin V Fluorescence Intensity
10 µM scramblase 1.5 x 107 cells/mL
3 h, pH 7.4, 37 C
41Inhibit Flippase
0.8
39
0.8
Normal
Number of Cells
2.2
80
2.1
NEM
FITC-Annexin V Fluorescence Intensity
10 µM scramblase 1.5 x 107 cells/mL
3 h, pH 7.4, 37 C
42PS Asymmetry
Blood coagulation Phagocytosis Aging Abnormal
adherence Chronic renal failure
OUTER
INNER
Cell signaling Enzyme activation Exocytosis Intrac
ellular fusion Mechanical stability
43Prothrombinase Assay
44Thrombin Hydrolysis Activity - Erythrocytes
NEM-pretreated
Normal
10 µM scramblase 1.5 x 107 cells/mL
3 h, pH 7.4, 37 C
45Conclusions
- Designed and characterized several series of
synthetic scramblases - Basic hydrogen-bonding principles
- Charge interactions
- Demonstrated effectiveness in both vesicle and
cellular systems
PS scramblase Ready to be tested for a variety
of biological responses AND ready to be used as a
research tool to study these biological responses
46Acknowledgements
Professor Bradley D. Smith Smith Group Members -
Past and Present Marty Deetz, Tim Lambert,
Ramesh Shukla, Atanas Koulov Castellino, Goodson,
and Welsh Labs University of Notre Dame,
NIH Family - Dad, Mom, Liz, Read, Jim, Lyn,
Jimmy, Thomas, Catie