Thermodynamic and kinetic stabilities of transmembrane helix bundles as revealed by single-pair FRET analysis: Effects of the number of membrane-spanning segments and cholesterol

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Table 1. Average lifetimes of monomers and dimers at 298 K

POPC

peptides

monomer lifetime

(ms)

POPC/cholesterol (7/3)

dimer lifetime

(ms)

monomer lifetime

(ms)

dimer lifetime

(ms)

----------------------------------------------------------------------------------------------------------------------------------------------1TM–1TM

not detected

not detected

562

261

1TM–2TM

545

99

159

381

2TM–2TM

1000

394

567

478

--------------------------------------------------------------------------------------------------------------------------------------------

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Table 2. Thermodynamic parameters of the association of helices in POPC membranes at 298 K

Peptides

∆Ga (kJ mol–1)

∆Ha (kJ mol–1)

–T∆Sa (kJ mol–1)

-------------------------------------------- -------------------------------------------- -----------------------------------------------POPC

+chol

difference

POPC

+chol

difference

POPC

+chol

difference

--------------------------------------------------------------------------------------------------------------------------------------------------------------1TM–1TM

–13.2 ± 0.2* –22.6 ± 0.1** –9.4

–23.7 ± 0.4* –84.1 ± 1.7** –60.4

+10.4 ± 0.4* +61.4 ± 1.7** +51.0

1TM–2TM

–19.3 ± 1.2

–28.3 ± 2.6

–9.0

–35.4 ± 1.0

–59.3 ± 3.2

+16.1 ± 1.0

2TM–2TM

–21.6 ± 2.2

–24.2 ± 3.1

–2.6

–54.7 ± 6.6

–109.1 ± 20–54.4

–23.9

+33.4 ± 6.6

+31.0 ± 3.2

+84.0 ± 20

+14.9

+50.6

--------------------------------------------------------------------------------------------------------------------------------------------------------------*data from ensemble measurements (assuming ∆Cp(a) = –0.5 J K–1 mol–1)25

**data from ensemble measurements (assuming ∆Cp(a) = 1.5 J K–1 mol–1)23

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Table 3. FTIR–PATR parameters for assessment of helix orientation at 298 K

POPC

Peptides

POPC/cholesterol(7/3)

α*

R*

(deg)

(deg)

------------------------------------------------------------------------------------------------------------------------------------------1TM–1TM

6.5 ± 0.6

1TM–2TM

4.8 ± 0.2

2TM–2TM

4.3 ± 0.1

~0

4.7 ± 0.2

17 ± 2

15 ± 3

3.6 ± 0.1

30 ± 1

22 ± 1

4.2 ± 0.1

23 ± 3

------------------------------------------------------------------------------------------------------------------------------------------* R and α indicate the dichroic ratio for the peptide amide I band and angle of helix orientation relative to the bilayer normal

calculated from R (Eq. 1), respectively.

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FIGURE LEGENDS

Figure 1. Single-pair FRET (sp-FRET) measurements. (a) Schematic illustration of a

surface-attached vesicle for sp-FRET imaging by total internal reflection microscopy. (b)

Representative time-courses of fluorescence intensities for Cy3B (green) and Cy5 (Red)

under excitation of Cy3B for 1TM–1TM (left column), 1TM–2TM (middle column), and

2TM–2TM pairs (right column) in POPC (upper row) and POPC/cholesterol (lower row) at

25°C. The number of analyzed vesicles is denoted by n. Only Cy3B fluoresced for 1TM–

1TM in POPC, indicating no association between the helices. Under other conditions, the

emissions fluctuated with an anticorrelation, reflecting the association–dissociation dynamics

of the helices. (c)(d) Photobleaching of Cy3B (c) and Cy5 (d) detected as a stepwise

decrease in the fluorescence intensities (arrowheads). The vesicles that had incorporated one

Cy3B-helix and one Cy5-helix were selected for analysis.

Figure 2. Sp-FRET analysis for 1TM–2TM and 2TM–2TM associations in POPC vesicles.

(a) HaMMy fitting for sp-FRET trajectories assuming two-state transitions. Black and green

lines indicate measured apparent FRET efficiency (Eapp) and the most probable fitting,

respectively. The monomer and dimer states correspond to Eapp values of ~0.4 and ~0.8,

respectively. (b) Histograms of natural logarithm of rate constants for dimer formation (kon)

and dimer dissociation (koff). The average ± error values were obtained from Gaussian

fitting of the histogram. (c) Histograms of Eapp.

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Figure 3. Sp-FRET analysis for 1TM–1TM, 1TM–2TM, and 2TM–2TM associations in

POPC/cholesterol (7/3) vesicles. (a) Histograms of the natural logarithm of rate constants for

dimer formation (kon) and dimer dissociation (koff). The average ± error values were

obtained from Gaussian fitting of the histogram. (b) Histograms of Eapp.

Figure 4. Temperature dependences of association free energy for (a) 1TM–2TM

associations and (b) 2TM–2TM associations. The temperature dependences were linearly

fitted to estimate the thermodynamic parameters by Eq. (7).

Figure 5. Amide region FTIR-PATR spectra for 1TM, 1TM/2TM(1/1) or 2TM incorporated

into membranes (peptides/lipids = 1/1000) at 25℃. The membrane films were hydrated with

D2O vapor. Red and black lines indicate raw spectra for the IR beam with its electric vector

parallel and perpendicular to the plane of incidence, respectively.

Figure 6. Effects of the membrane lateral pressure profile on the shape of the transmembrane

helix bundle. Because of the small headgroup of cholesterol, the headgroup and hydrocarbon

core regions have lower and higher lateral pressures, respectively, in cholesterol-containing

membranes (red arrows), compared with in pure POPC membranes. POPC membranes

stabilize helix bundles without helix tilt (parallel helix bundle). On the other hand,

cholesterol-containing membranes can stabilize helix bundles with significant helix tilt

(hourglass-shaped helix bundle).

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Figure 7. Alterations in the helix macrodipole interactions upon the formation of the 1TM–

2TM bundle (a) and 2TM–2TM bundle (b). Upward and downward arrows in the circles

indicate C-terminus-up and C-terminus-down transmembrane topologies of the helices,

respectively. Antiparallel (A) and parallel (P or P’) interhelical contacts are shown as blue

and red lines, respectively. (a) 1TM–2TM association generates A + P interactions, which is

zero assuming symmetric packing of the helices in the bundle. (b) The repulsive interactions

in 2TM–2TM bundle are minimized in square packing (P’). The association generates 2A +

2P’ interactions, which have a negative ∆H value.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Supporting information

Thermodynamic and kinetic stabilities of transmembrane helix bundles as

revealed by single-pair FRET analysis: Effects of the number of membranespanning segments and cholesterol

Yoshiaki Yano, Yuta Watanabe, and Katsumi Matsuzaki

NBD

Cy3B

Cy5

peptide

peptide

peptide

Figure S1. Structures of florescent probes used in this study.

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Figure S2. HPLC chromatograms of transmembrane peptides. Purified fluorophore-labeled

peptides (I–VI) were analyzed with a PLRP-s analytical column (150×4.6 mm) at 50°C. The 1TM

peptides were eluted with a linear gradient of H2O/0.1% TFA and AcCN/0.1% TFA from 65 to 95%

(I), and from 10 to 90% (II, III). The 2TM peptides (IV, V, VI) were eluted with a linear gradient of

formic acid/H2O (2/3, v/v) and formic acid/2-propanol (4/1, v/v) from 50 to 80% (0–5min) and 80 to

95% (5–30 min).

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