FLUORESCENCE SPECTROSCOPY

Tryptophan fluorescence emission was measured using a Tecan infinite M1000 pro plate reader. Excitation was 280 nm and emission was recorded in the range 300 - 450 nm using steps of 1 nm and bandwidths of 5 and 10 nm for excitation and emission. For each measurement 3 scans at 25 °C were averaged and corrected for scattering. Black Greiner 96 well plates were used.

For experiments with increasing lipid to peptide ratio mixtures of peptides at constant concentrations of tryptophan containing peptides [X_GW]  =  2.5 µM and increasing concentrations of lipid up to 5 mM were prepared with constant

96

Chapter IV

volume of 200 µl. The complexes E_GW/K and E/K_GW where used at molar ratios 1 : 1 with [X_GW] = 2.5 µM. For determination of the highest partition coefficient of [K_GW] additional data points were collected with lipid concentrations up to 1.25 mM. The maxima of the emission λ_max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40-42 _{The fluorescence intensities F in}

presence of vesicles were used to calculate the fraction of membrane bound peptide P_b by:

16 CHAPTER IV

determination of the highest partition coefficient of [KGW] additional data points were collected

with lipid concentrations up to 1.25 mM. The maxima of the emission max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40- 42 _{The fluorescence intensities F in presence of vesicles were used to calculate the fraction of}

membrane bound peptide Pb by:

𝑃𝑃𝑏𝑏= [𝑋𝑋𝐺𝐺𝐺𝐺]_𝐹𝐹𝐹𝐹 − 𝐹𝐹0

∞− 𝐹𝐹0 (1)

with the concentration of the tryptophan containing peptide [XGW], F0the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound 𝐹𝐹∞ was

obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1/[Lipid] = 0. To obtain the partition coefficient Kp the data was linearly fitted to:

𝑋𝑋𝑏𝑏∗= 𝐾𝐾𝑝𝑝𝐶𝐶𝑓𝑓 (2)

where Xb* is the molar ratio of bound peptide per accessible (outer leaflet ~60%) lipid and Cf the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid]:[XGW] = 1000:1,

were mixed with increasing concentrations of quencher yielding constant concentrations [XGW]

= 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching

experiments with LPKGW and LPEGW concentrations were [LPXGW] = 2.5 µM and

[Lipid]:[LPXGW]=100:1. The fluorescence emission in presence F and in absence F0 of quencher was fitted to yield the Stern-Vollmer constants KSV by:

𝐹𝐹0

𝐹𝐹 = 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] + 1. (3)

For quenching with brominated lipids vesicles were composed of DOPC:diBr- PC:DOPE:Cholesterol 1:1:1:1, for the fluorescence intensity in absence of quencher F0 DOPC:DOPE:Cholesterol 2:1:1 was used. Experiments were done at [LPXGW] = 10 µM,

[Lipid]:[LPXGW]=100:1 and [XGW] = 2.5 µM [Lipid]:[XGW]=1000:1. The quenching efficiency

QE was calculated by:

𝑄𝑄𝑄𝑄 = (1 −_𝐹𝐹𝐹𝐹

0) × 100%. (4)

To estimate the distance of the fluorophore from the center of the bilayer Zcf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower F1 and deeper}

quencher F2: 𝑍𝑍𝑐𝑐𝑓𝑓= − 1_{𝜋𝜋𝐶𝐶 ln (}𝐹𝐹1 𝐹𝐹2) − 𝐿𝐿21 2𝐿𝐿21 + 𝐿𝐿𝑐𝑐1 (5)

with the concentration of the tryptophan containing peptide [X_GW], F₀ the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound was obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1 / [Lipid]  =  0. To obtain the partition coefficient K_p the data was linearly fitted to:

16 CHAPTER IV

determination of the highest partition coefficient of [KGW] additional data points were collected

with lipid concentrations up to 1.25 mM. The maxima of the emission max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40- 42 _{The fluorescence intensities F in presence of vesicles were used to calculate the fraction of}

membrane bound peptide Pb by:

𝑃𝑃𝑏𝑏= [𝑋𝑋𝐺𝐺𝐺𝐺]_𝐹𝐹𝐹𝐹 − 𝐹𝐹0

∞− 𝐹𝐹0 (1)

with the concentration of the tryptophan containing peptide [XGW], F0the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound 𝐹𝐹∞ was

obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1/[Lipid] = 0. To obtain the partition coefficient Kp the data was linearly fitted to:

𝑋𝑋𝑏𝑏∗= 𝐾𝐾𝑝𝑝𝐶𝐶𝑓𝑓 (2)

where Xb* is the molar ratio of bound peptide per accessible (outer leaflet ~60%) lipid and Cf the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid]:[XGW] = 1000:1,

were mixed with increasing concentrations of quencher yielding constant concentrations [XGW]

= 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching

experiments with LPKGW and LPEGW concentrations were [LPXGW] = 2.5 µM and

[Lipid]:[LPXGW]=100:1. The fluorescence emission in presence F and in absence F0 of quencher was fitted to yield the Stern-Vollmer constants KSV by:

𝐹𝐹0

𝐹𝐹 = 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] + 1. (3)

For quenching with brominated lipids vesicles were composed of DOPC:diBr- PC:DOPE:Cholesterol 1:1:1:1, for the fluorescence intensity in absence of quencher F0 DOPC:DOPE:Cholesterol 2:1:1 was used. Experiments were done at [LPXGW] = 10 µM,

[Lipid]:[LPXGW]=100:1 and [XGW] = 2.5 µM [Lipid]:[XGW]=1000:1. The quenching efficiency

QE was calculated by:

𝑄𝑄𝑄𝑄 = (1 −_𝐹𝐹𝐹𝐹

0) × 100%. (4)

To estimate the distance of the fluorophore from the center of the bilayer Zcf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower F}

1 and deeper quencher F2: 𝑍𝑍𝑐𝑐𝑓𝑓= − 1_{𝜋𝜋𝐶𝐶 ln (}𝐹𝐹1 𝐹𝐹2) − 𝐿𝐿21 2𝐿𝐿21 + 𝐿𝐿𝑐𝑐1 (5)

where X_b* _{is the molar ratio of bound peptide per accessible (outer leaflet ~60%)} lipid and C_f the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid] : [X_GW] = 1000:1, were mixed with increasing concentrations of quencher yielding constant concentrations [X_GW] = 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching experiments with LPK_GW and LPE_GW concentrations were [LPX_GW] = 2.5 µM and [Lipid] : [LPX_GW] = 100:1. The fluorescence emission in presence F and in absence F₀ of quencher was fitted

to yield the Stern-Vollmer constants K_SV by:

16 CHAPTER IV

determination of the highest partition coefficient of [KGW] additional data points were collected

with lipid concentrations up to 1.25 mM. The maxima of the emission max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40- 42 _{The fluorescence intensities F in presence of vesicles were used to calculate the fraction of}

membrane bound peptide Pb by:

𝑃𝑃𝑏𝑏= [𝑋𝑋𝐺𝐺𝐺𝐺]_𝐹𝐹𝐹𝐹 − 𝐹𝐹0

∞− 𝐹𝐹0 (1)

with the concentration of the tryptophan containing peptide [XGW], F0the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound 𝐹𝐹∞ was

obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1/[Lipid] = 0. To obtain the partition coefficient Kp the data was linearly fitted to:

𝑋𝑋𝑏𝑏∗= 𝐾𝐾𝑝𝑝𝐶𝐶𝑓𝑓 (2)

where Xb* is the molar ratio of bound peptide per accessible (outer leaflet ~60%) lipid and Cf the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid]:[XGW] = 1000:1,

were mixed with increasing concentrations of quencher yielding constant concentrations [XGW]

= 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching

experiments with LPKGW and LPEGW concentrations were [LPXGW] = 2.5 µM and

[Lipid]:[LPXGW]=100:1. The fluorescence emission in presence F and in absence F0 of quencher was fitted to yield the Stern-Vollmer constants KSV by:

𝐹𝐹0

𝐹𝐹 = 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] + 1. (3)

For quenching with brominated lipids vesicles were composed of DOPC:diBr- PC:DOPE:Cholesterol 1:1:1:1, for the fluorescence intensity in absence of quencher F0 DOPC:DOPE:Cholesterol 2:1:1 was used. Experiments were done at [LPXGW] = 10 µM,

[Lipid]:[LPXGW]=100:1 and [XGW] = 2.5 µM [Lipid]:[XGW]=1000:1. The quenching efficiency

QE was calculated by:

𝑄𝑄𝑄𝑄 = (1 −_𝐹𝐹𝐹𝐹

0) × 100%. (4)

To estimate the distance of the fluorophore from the center of the bilayer Zcf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower F}

1 and deeper quencher F2: 𝑍𝑍𝑐𝑐𝑓𝑓= − 1_{𝜋𝜋𝐶𝐶 ln (}𝐹𝐹1 𝐹𝐹2) − 𝐿𝐿21 2𝐿𝐿21 + 𝐿𝐿𝑐𝑐1 (5)

For quenching with brominated lipids vesicles were composed of DOPE  :  diBr-PC:DOPE : Cholesterol 1 : 1 : 1 : 1, for the fluorescence intensity in absence of quencher F₀DOPE : DOPE : Cholesterol 2 : 1 : 1 was used. Experiments were done at [LPX_GW] = 10 µM, [Lipid]:[LPX_GW] = 100:1 and [X_GW] = 2.5 µM [Lipid] : [X_GW] = 1000 : 1. The quenching efficiency QE was calculated by:

16 CHAPTER IV

determination of the highest partition coefficient of [KGW] additional data points were collected

with lipid concentrations up to 1.25 mM. The maxima of the emission max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40- 42 _{The fluorescence intensities F in presence of vesicles were used to calculate the fraction of}

membrane bound peptide Pb by:

𝑃𝑃𝑏𝑏= [𝑋𝑋𝐺𝐺𝐺𝐺]_𝐹𝐹𝐹𝐹 − 𝐹𝐹0

∞− 𝐹𝐹0 (1)

with the concentration of the tryptophan containing peptide [XGW], F0the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound 𝐹𝐹∞ was

obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1/[Lipid] = 0. To obtain the partition coefficient Kp the data was linearly fitted to:

𝑋𝑋𝑏𝑏∗= 𝐾𝐾𝑝𝑝𝐶𝐶𝑓𝑓 (2)

where Xb* is the molar ratio of bound peptide per accessible (outer leaflet ~60%) lipid and Cf the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid]:[XGW] = 1000:1,

were mixed with increasing concentrations of quencher yielding constant concentrations [XGW]

= 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching

experiments with LPKGW and LPEGW concentrations were [LPXGW] = 2.5 µM and

[Lipid]:[LPXGW]=100:1. The fluorescence emission in presence F and in absence F0 of quencher was fitted to yield the Stern-Vollmer constants KSV by:

𝐹𝐹0

𝐹𝐹 = 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] + 1. (3)

For quenching with brominated lipids vesicles were composed of DOPC:diBr- PC:DOPE:Cholesterol 1:1:1:1, for the fluorescence intensity in absence of quencher F0 DOPC:DOPE:Cholesterol 2:1:1 was used. Experiments were done at [LPXGW] = 10 µM,

[Lipid]:[LPXGW]=100:1 and [XGW] = 2.5 µM [Lipid]:[XGW]=1000:1. The quenching efficiency

QE was calculated by:

𝑄𝑄𝑄𝑄 = (1 −_𝐹𝐹𝐹𝐹

0) × 100%. (4)

To estimate the distance of the fluorophore from the center of the bilayer Zcf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower F1 and deeper}

quencher F2: 𝑍𝑍𝑐𝑐𝑓𝑓= − 1_{𝜋𝜋𝐶𝐶 ln (}𝐹𝐹1 𝐹𝐹2) − 𝐿𝐿21 2𝐿𝐿21 + 𝐿𝐿𝑐𝑐1 (5)

To estimate the distance of the fluorophore from the center of the bilayer Z_cf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower} F₁ and deeper quencher F₂:

97

Chapter IV

16 CHAPTER IV

determination of the highest partition coefficient of [KGW] additional data points were collected

with lipid concentrations up to 1.25 mM. The maxima of the emission max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium.26,40- 42 _{The fluorescence intensities F in presence of vesicles were used to calculate the fraction of}

membrane bound peptide Pb by:

𝑃𝑃𝑏𝑏= [𝑋𝑋𝐺𝐺𝐺𝐺]_𝐹𝐹𝐹𝐹 − 𝐹𝐹0

∞− 𝐹𝐹0 (1)

with the concentration of the tryptophan containing peptide [XGW], F0the fluorescence intensity without vesicles. The fluorescence intensity when all peptide is membrane bound 𝐹𝐹∞ was

obtained from extrapolation of a double reciprocal plot of F vs the lipid concentration [Lipid] to 1/[Lipid] = 0. To obtain the partition coefficient Kp the data was linearly fitted to:

𝑋𝑋𝑏𝑏∗= 𝐾𝐾𝑝𝑝𝐶𝐶𝑓𝑓 (2)

where Xb* is the molar ratio of bound peptide per accessible (outer leaflet ~60%) lipid and Cf the concentration of free (unbound) peptide.

For acrylamide quenching, peptides in absence or presence of vesicles [Lipid]:[XGW] = 1000:1,

were mixed with increasing concentrations of quencher yielding constant concentrations [XGW]

= 2.5 µM and V = 200 µl and increasing [Acrylamide] = 0…150 µM in each well. In quenching

experiments with LPKGW and LPEGW concentrations were [LPXGW] = 2.5 µM and

[Lipid]:[LPXGW]=100:1. The fluorescence emission in presence F and in absence F0 of quencher was fitted to yield the Stern-Vollmer constants KSV by:

𝐹𝐹0

𝐹𝐹 = 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] + 1. (3)

For quenching with brominated lipids vesicles were composed of DOPC:diBr- PC:DOPE:Cholesterol 1:1:1:1, for the fluorescence intensity in absence of quencher F0 DOPC:DOPE:Cholesterol 2:1:1 was used. Experiments were done at [LPXGW] = 10 µM,

[Lipid]:[LPXGW]=100:1 and [XGW] = 2.5 µM [Lipid]:[XGW]=1000:1. The quenching efficiency

QE was calculated by:

𝑄𝑄𝑄𝑄 = (1 −_𝐹𝐹𝐹𝐹

0) × 100%. (4)

To estimate the distance of the fluorophore from the center of the bilayer Zcf the parallax method26,28,32,33 _{was applied using the fluorescence intensities of shallower F1 and deeper}

quencher F2: 𝑍𝑍𝑐𝑐𝑓𝑓= − 1_{𝜋𝜋𝐶𝐶 ln (}𝐹𝐹1 𝐹𝐹2) − 𝐿𝐿21 2𝐿𝐿21 + 𝐿𝐿𝑐𝑐1 (5)

where C is the molar fraction per lipid area (~60 Å2₎31_{, L}

21 is the distance between deeper and shallower quencher, and L_c1 is the distance between shallow quencher and bilayer center. For PM the fluorescence intensities of the two closest quenchers were used only. For application of the distribution analysis method (DA), the quenching profile QP as function of the distance to the bilayer center h was fitted by two symmetric Gaussians:

CHAPTER II 17

where C is the molar fraction per lipid area (~60 Å2₎31_{, L}

21 is the distance between deeper and shallower quencher, and Lc1 is the distance between shallow quencher and bilayer center. For PM the fluorescence intensities of the two closest quenchers were used only. For application of the distribution analysis method (DA), the quenching profile QP as function of the distance to the bilayer center h was fitted by two symmetric Gaussians:

𝑄𝑄𝑄𝑄 =𝐹𝐹_{𝐹𝐹 − 1 =}0 𝑆𝑆 𝜎𝜎√2𝜋𝜋𝑒𝑒 −(ℎ−ℎ𝑚𝑚)2 2𝜎𝜎2 ₊ 𝑆𝑆 𝜎𝜎√2𝜋𝜋𝑒𝑒 −(ℎ+ℎ𝑚𝑚)2 2𝜎𝜎2 (6)

the parameter hm stands for the most probable distance between fluorophore and bilayer center,

S is the areas and  the dispersions of the Gaussians. Due to the limited amount of data values of hm were fixed at different values, while leaving S and  vary freely.

Circular dichroism spectroscopy

CD spectra were measured using a Jasco J815 CD spectrometer equipped with a Jasco PTC 123 peltier temperature controller. Quartz cuvettes with pathlengths l of 1, 2 and 5 mm were used. Peptide stock solutions were diluted with buffer to the desired total peptide concentrations and eventually mixed with vesicles at [Lipid]:[Peptide]=100:1 at 5 µM total peptide concentration. Spectra of the lipopeptides tethered to vesicle were measured at [Lipid]:[LPXGW]=100:1 in 250 µM total lipid concentration and complementary peptide E or

K was added

[LPXGW]:[YGW ]=1:1. Spectra were baseline corrected and the mean residue ellipticity [] was

calculated by:

[𝜃𝜃] =_{𝑙𝑙 𝑐𝑐}𝜃𝜃

𝑀𝑀 𝑁𝑁 (7)

where  is the observed ellipticity, l the pathlength, cM the molar total peptide concentration and N the number of amino acids per peptide. The relative  - helicity rh was calculated from the ellipticity at 222nm []222nm by:21,22

𝑟𝑟ℎ = [𝜃𝜃]222𝑛𝑛𝑛𝑛

−40 103_degcm2_dmol−1 _{(1 − 4.6}

𝑁𝑁 )

100% ₍₈₎

For temperature dependent unfolding experiments peptide stock solutions were diluted with buffer to the desired total peptide concentrations in the range 6 – 50 µM. []222nm was measured

as a function of temperature T in a 2 mm quartz cuvette at a heating rate of 40 °C/h in the range 2 – 95 °C. Additionally, CD spectra between 190 - 260 nm were collected at T = 5, 25 and 80 °C. Spectra taken at 5 °C before and directly after a full heating cycle were found to be fully

the parameter h_m stands for the most probable distance between fluorophore and bilayer center, S is the areas and σ the dispersions of the Gaussians. Due to the limited amount of data values of h_m were fixed at different values, while leaving S and σ vary freely.

CIRCULAR DICHROISM SPECTROSCOPY

CD spectra were measured using a Jasco J815 CD spectrometer equipped with a Jasco PTC 123 peltier temperature controller. Quartz cuvettes with pathlengths l of 1, 2 and 5 mm were used. Peptide stock solutions were diluted with buffer to the desired total peptide concentrations and eventually mixed with vesicles at [Lipid] : [Peptide] = 100 : 1 at 5 µM total peptide concentration. Spectra of the lipopeptides tethered to vesicle were measured at [Lipid] : [LPX_GW]  =  100 : 1 in 250 µM total lipid concentration and complementary peptide E or K was added [LPX_GW] : [Y_GW ]  =  1 : 1. Spectra were baseline corrected and the mean residue ellipticity [θ] was calculated by:

CHAPTER II 17

where C is the molar fraction per lipid area (~60 Å2₎31_{, L21 is the distance between deeper and}

shallower quencher, and Lc1 is the distance between shallow quencher and bilayer center. For PM the fluorescence intensities of the two closest quenchers were used only. For application of the distribution analysis method (DA), the quenching profile QP as function of the distance to the bilayer center h was fitted by two symmetric Gaussians:

𝑄𝑄𝑄𝑄 =𝐹𝐹_{𝐹𝐹 − 1 =}0 𝑆𝑆 𝜎𝜎√2𝜋𝜋𝑒𝑒

−(ℎ−ℎ_2𝜎𝜎𝑚𝑚₂)2₊ 𝑆𝑆

𝜎𝜎√2𝜋𝜋𝑒𝑒

−(ℎ+ℎ_2𝜎𝜎𝑚𝑚₂)2 ₍₆₎

the parameter hm stands for the most probable distance between fluorophore and bilayer center,

S is the areas and  the dispersions of the Gaussians. Due to the limited amount of data values of hm were fixed at different values, while leaving S and  vary freely.

Circular dichroism spectroscopy

CD spectra were measured using a Jasco J815 CD spectrometer equipped with a Jasco PTC 123 peltier temperature controller. Quartz cuvettes with pathlengths l of 1, 2 and 5 mm were used. Peptide stock solutions were diluted with buffer to the desired total peptide concentrations and eventually mixed with vesicles at [Lipid]:[Peptide]=100:1 at 5 µM total peptide concentration. Spectra of the lipopeptides tethered to vesicle were measured at [Lipid]:[LPXGW]=100:1 in 250 µM total lipid concentration and complementary peptide E or

K was added

[LPXGW]:[YGW ]=1:1. Spectra were baseline corrected and the mean residue ellipticity [] was

calculated by:

[𝜃𝜃] =_{𝑙𝑙 𝑐𝑐}𝜃𝜃

𝑀𝑀 𝑁𝑁 (7)

where  is the observed ellipticity, l the pathlength, cM the molar total peptide concentration and N the number of amino acids per peptide. The relative  - helicity rh was calculated from the ellipticity at 222nm []222nm by:21,22

𝑟𝑟ℎ = [𝜃𝜃]222𝑛𝑛𝑛𝑛

−40 103_degcm2_dmol−1 _{(1 − 4.6}

𝑁𝑁 )

100% ₍₈₎

For temperature dependent unfolding experiments peptide stock solutions were diluted with buffer to the desired total peptide concentrations in the range 6 – 50 µM. []222nm was measured

as a function of temperature T in a 2 mm quartz cuvette at a heating rate of 40 °C/h in the range 2 – 95 °C. Additionally, CD spectra between 190 - 260 nm were collected at T = 5, 25 and 80 °C. Spectra taken at 5 °C before and directly after a full heating cycle were found to be fully

where θ is the observed ellipticity, l the pathlength, c_M the molar total peptide concentration and N the number of amino acids per peptide. The relative α - helicity rh was calculated from the ellipticity at 222nm [θ]_222nm by:21,22

CHAPTER II 17

where C is the molar fraction per lipid area (~60 Å2₎31_{, L21 is the distance between deeper and}

shallower quencher, and Lc1 is the distance between shallow quencher and bilayer center. For PM the fluorescence intensities of the two closest quenchers were used only. For application of the distribution analysis method (DA), the quenching profile QP as function of the distance to the bilayer center h was fitted by two symmetric Gaussians:

𝑄𝑄𝑄𝑄 =𝐹𝐹_{𝐹𝐹 − 1 =}0 𝑆𝑆 𝜎𝜎√2𝜋𝜋𝑒𝑒 −(ℎ−ℎ𝑚𝑚)2 2𝜎𝜎2 ₊ 𝑆𝑆 𝜎𝜎√2𝜋𝜋𝑒𝑒 −(ℎ+ℎ𝑚𝑚)2 2𝜎𝜎2 ₍₆₎

the parameter hm stands for the most probable distance between fluorophore and bilayer center,

S is the areas and  the dispersions of the Gaussians. Due to the limited amount of data values of hm were fixed at different values, while leaving S and  vary freely.

Circular dichroism spectroscopy

CD spectra were measured using a Jasco J815 CD spectrometer equipped with a Jasco PTC 123 peltier temperature controller. Quartz cuvettes with pathlengths l of 1, 2 and 5 mm were used. Peptide stock solutions were diluted with buffer to the desired total peptide concentrations and eventually mixed with vesicles at [Lipid]:[Peptide]=100:1 at 5 µM total

In document Coiled-coils on lipid membranes : a new perspective on membrane fusion (Page 96-102)