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Facilitated Phospholipid FlipFlop Across Bilayer Membranes

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Sulfonamide (2100 80 M-1) Amide (30 4 M-1) Ester ( 1 M-1) Crystal Structures ... Methyl Ester Version. Dr. T. Lambert. Erythrocyte Morphology Study ... – PowerPoint PPT presentation

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Title: Facilitated Phospholipid FlipFlop Across Bilayer Membranes


1
Facilitated Phospholipid Flip-Flop Across Bilayer
Membranes J. Middleton Boon Department of
Chemistry and Biochemistry University of Notre
Dame April 9, 2003
2
Flip-Flop
Boon and Smith, Curr. Opin. Chem. Biol. 2002, 6,
749-756.
3
SM
PC
PS
PE
Op den Kamp, Annu. Rev. Biochem. 1979, 48,
47-71. Boon and Smith, Med. Res. Rev. 2002, 22,
251-281.
4
flippase
Zwaal and Schroit, Blood 1997, 89, 1121-1132.
5
SM
PC
PS
PE
  • I. PC Scramblases
  • 1) TREN-Derived
  • 2) Steroid-Derived
  • II. PE Scramblases
  • III. PS Scramblases

6
SM
PC
PS
PE
I. PC Scramblases 1) TREN-Derived 2)
Steroid-Derived
7
1) TREN-Derived Scramblases
Boon and Smith, J. Am. Chem. Soc. 1999, 121,
11924-11925. Boon, et al., J. Org. Chem. 2001,
67, 2168-2174.
8
Flip Assay
Add dithionite
Add detergent
POPC
NBD-X
POPC (25 µM)/NBD-X (0.5)/pH 7.4, 25 C
9
NBD-PC Flip Across Vesicle Membranes
POPC 25 µM (0.5 NBD-PC)
Scramblase 38 µM
10
POPC Binding
H2O
X

H2O
X
H2O
X
NH
CDCl3, 25 C
11
Titration Isotherms
CDCl3, 25 C POPC
Sulfonamide (2100 80 M-1) gt Amide (30 4 M-1)
gt Ester (lt1 M-1)
12
Crystal Structures
Sulfonamide
Amide
Dr. S. Brown
13
pKa Measurements
991 CD3OD/D2O, 25 C
Sulfonamide 4.00 0.01 Amide 5.52 0.02
Ester 3.75 0.01
14
Membrane Interactions
Rh emission
Dr. T. Lambert
NBD emission/ Rh excitation
NBD excitation
POPC 25 µM (0.6 Rh-PE)
NBD-sulfonamide 38 µM
15
Add detergent
Add NBD-sulf
POPC 25 µM (0.6 Rh-PE)
NBD-sulfonamide 38 µM
16
Membrane Partitioning
Dr. T. Lambert
? POPC (25 µM)/Dansyl-sulfonamide (5 mol )
17
Translocation 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
18
Erythrocyte 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.
20
Sulfonamide
Amide
Scramblase 35 µM 2 h, pH 7.4, 37 C
Ester
21
0
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.
22
Morphological Index Curves
Scramblase 35 µM
pH 7.4, 37 C
Average of 3 separate experiments
23
NBD-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
24
Peroxisome 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.
25
2) Second Generation Steroid-Derived Scramblases
Dr. T. Lambert
Lambert, Boon, et al., J. Am. Chem. Soc. 2002,
124, 5276-5277.
26
NBD-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
27
UV Binding Study
991 CHCl3/CH3OH, 25 C POPC (1.2
0.1) x 105 M-1
28
Methyl Ester Version
Dr. T. Lambert
29
Erythrocyte Morphology Study
Scramblase 40 µM
pH 7.4, 37 C
Average of 3 separate experiments
30
NBD-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
31
II. PE Scramblases
SM
PC
PS
PE
Boon, et al., Chem. Commun. 2002, 260-261.
32
NBD-Lipid Flip Across Vesicle Membranes
Dr. R. Shukla
POPC 25 µM (0.5 NBD-X)
Scramblase 100 µM
33
NBD-Lipid Flip Across Vesicle Membranes
Dr. P. Scrimin
POPC 25 µM (0.5 NBD-X) Scramblase
8 µM Average of 3 separate
experiments
34
III. PS Scramblases
SM
PC
PS
PE
Boon, et al., J. Am. Chem. Soc. 2003, submitted.
35
NBD-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
36
Half-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
37
Translocation Mechanism
38
Binding of Annexin V-FITC
39
Flow 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
40
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
41
Inhibit 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
42
PS Asymmetry
Blood coagulation Phagocytosis Aging Abnormal
adherence Chronic renal failure
OUTER
INNER
Cell signaling Enzyme activation Exocytosis Intrac
ellular fusion Mechanical stability
43
Prothrombinase Assay
44
Thrombin Hydrolysis Activity - Erythrocytes
NEM-pretreated
Normal
10 µM scramblase 1.5 x 107 cells/mL
3 h, pH 7.4, 37 C
45
Conclusions
  • 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
46
Acknowledgements
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
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