unfolding dynamics of cytochrome c revealed by single … · 2011-02-07 · unfolding dynamics of...

Supplementary Information

Unfolding Dynamics of Cytochrome c Revealed by Single-Molecule and

Ensemble-Averaged Spectroscopy

Jungkweon Choi, Sooyeon Kim, Takashi Tachikawa, Mamoru Fujitsuka, and Tetsuro Majima*

MALDI-TOF mass spectrum of Apocytochrome c

12095.070

apoCyt-C 0:B11 MS R aw

0

500

1000

1500

Inte

ns.

[a.u

.]

12707.680

Cyt-C 0:B13 MS R aw

0

1000

2000

3000

4000

5000

Inte

ns.

[a.u

.]

5000 10000 15000 20000 25000m /z

Fig. S1. Mass spectra of ApoCytc (upper) and WT Cytc (lower). Fluorescence lifetimes of Cytc-A488 and Alexa 488 with various concentrations of GdHCl (Bulk

experiments)

All the decay profiles measured from Alexa 488 can be fitted by a single exponential function

with relaxation times of 3.8 ± 0.1 ns (see Fig. S3b). In contrast, all fluorescence decay profiles

measured from Cytc-A488 at various GdHCl concentrations can be expressed by a bi-exponential

function. By a simple analysis of all the fluorescence decay profiles, we found three components

with different fluorescence lifetimes in the GdHCl-induced unfolding reaction of Cytc-A488.

Thus, we fitted all the decay profiles of Cytc-A488 with a tri-exponential function. A global

fitting analysis revealed three relaxation times of 0.36, 0.81, and 3.21 ns in the GdHCl-induced

unfolding reaction of Cytc-A488.

# Supplementary Material (ESI) for Physical Chemistry Chemical Physics# This journal is (c) The Owner Societies 2011

1200

1000

800

600

400

200

0

20181614121086420

time / ns Fig. S2. Global fitting results of the fluorescence decay profiles of Cytc-A488 as a function of the concentration of GdHCl. All decay profiles of Cytc-A488 were expressed by a tri-exponential function with relaxation times of 0.36, 0.81, and 3.21 ns. The theoretical fits obtained from the fitting analysis are shown in red.

500 520 540 560 580 600 620 6400

20

40

60

80

100

120

140

Fl.

Inte

ns

ity

/a.u

.

Wavelength / nm

Alexa Fluor 488 in 0.0M GdHCl Alexa Fluor 488 in 1.0M GdHCl Alexa Fluor 488 in 2.0M GdHCl Alexa Fluor 488 in 3.2M GdHCl Alexa Fluor 488 in 5.0M GdHCl Alexa Fluor 488 in 8.0M GdHCl

4 6 8 10 12 14 16 18 20 22 241

10

100

1000

Fl.

Inte

ns

ity

/ a.u

.

Time / ns

0.0M GdHCl 0.5M GdHCl 1.0M GdHCl 2.0M GdHCl 3.2M GdHCl 4.0M GdHCl

a) b)

Fig. S3. a) Fluorescence spectra of Alexa 488 as a function of the concentration of GdHCl in 100 mM phosphate buffer. b) The fluorescence decay profiles of Alexa 488 as a function of the concentration of GdHCl in 100 mM phosphate buffer (pH 7.0). Transient absorption measurement

The sub-picosecond transient absorption spectra were measured by the pump and probe

method using a regeneratively amplified titanium sapphire laser (Spectra Physics, Spitfire Pro F, 1

kHz) pumped by a Nd:YLF laser (Spectra Physics, Empower 15) for an aqueous solution containing

Cytc-A488 or Alexa 488 in 100 mM Na phosphate buffer (pH 7.0). The seed pulse was generated by

the titanium sapphire laser (Spectra Physics, Tsunami 3941-M1BB, full width at half-maximum 80 fs,


800 nm). An excitation pulse at 485 nm was generated by optical parametric amplifier (Spectra

Physics, OPA-800CF). A white continuum pulse, which was generated by focusing the residual of the

fundamental light to a flowing water cell after a computer controlled optical delay, was divided into

two parts and used as the probe and the reference lights, of which the latter was used to compensate

the laser fluctuation. Both probe and reference lights were directed to a rotating sample cell with 1.0

mm of optical path and were detected with a charge-coupled device detector equipped with a

polychromator (Solar, MS3504). The pump pulse was chopped by a mechanical chopper synchronized

to one-half of the laser repetition rate, resulting in a pair of the spectra with and without the pump,

from which an absorption change induced by the pump pulse was estimated.

By the femtosecond transient absorption measurement, we found that the transient

absorption spectra of Cytc-A488 obtained after 485 nm laser excitation without GdHCl are similar

to that of Alexa 488. These spectra display the transient absorption signal at around 430 nm and

the broad bleaching signal due to the absorption band and emission of Alexa 488, respectively.

The transient signal of Cytc-A488 observed at 430 nm certainly originates from the formation and

decay of the excited singlet state of Alexa 488. However, we could not observe any dynamics due

to energy transfer between Alexa 488 and heme, as shown in Fig. 4a and S4.

On the other hand, we have to consider photoinduced electron transfer (PET) between

Alexa 488 to the iron ion of the heme in Cytc-A488. As shown in Fig. S6, the absorption band of

the reduced Cytc is very different with that of the oxidized Cytc. Thus, if PET from Alexa 488 to

the iron ion of the heme occurs in this system, the transient absorption band of the reduced Cytc

generated by PET should be observed. However, we could not observe any signal related to PET

between Alexa 488 and heme. This result means that PET from Alexa 488 to the iron ion of the

heme does not take place in this system. Thus, we conclude that the fluorescence quenching

observed from the folded Cytc-A488 is mainly due to PET between electron-donating amino acids

such as tryptophan (Trp) and a dye attached to a protein rather than the energy transfer.

560 600 640 680 720 760-0.020

-0.018

-0.016

-0.014

-0.012

-0.010

-0.008

-0.006

-0.004

-0.002

0.000

0.002

O

D

Wavelength / nm

at 0 ps at 10 ps at 50 ps at 100 ps at 200 ps at 500 ps at 1000 ps at 1500 ps

Fig. S4. Transient absorption spectra of Cytc-A488 without GdHCl obtained after 485 nm laser excitation in 100 mM phosphate buffer.


Fig. S5. Transient absorption spectra of WT Cytc without GdHCl after 360 nm laser excitation in 100 mM phosphate buffer.

Fig. S6. Absorption spectra of the oxidized and reduced wide-type Cytc without GdHCl in 100 mM phosphate buffer.

Fluorescence quenching of Alexa 488 by the addition of Trp

Fig. S7 shows that the fluorescence spectra of Alexa 488 as a function of the concentration of

Trytophan (Trp) in 100 mM phosphate buffer. The fluorescence intensities of Alexa 488 were

significantly decreased by increasing the concentration of Trp. The quenching constant kq is

determined from the Stern-Volmer plot as follows:

0

0

1 [Q]SV

SV q

FK

FK k

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively.

[Q] is the concentration of the quencher (Trp). 0 is the lifetime of the fluorophore in the absence of

quencher.

420 440 460 480 500 520 540 560 580 600 620 640 660-0.005

0.000

0.005

0.010

0.015

O

D

Wavelength / nm

at 6 ps at 42 ps

300 350 400 450 500 550 6000.0

0.2

0.4

0.6

0.8

1.0

1.2

Ab

sorb

ance

Wavelength / nm

WT Fe2+-Cytc

WT Fe3+-Cytc


0.000 0.005 0.010 0.015 0.020 0.025 0.030

1.0

1.2

1.4

1.6

1.8

2.0

F0/F

[Trp] / M

K (Slope) = 27.3 M-1

500 520 540 560 580 600 620 6400

5

10

15

20

25

Fl.

Inte

nsi

ty /

a.u

.

Wavelength / nm

Trp 0 mM Trp 5 mM Trp 10 mM Trp 15 mM Trp 20 mM Trp 30 mM

b)a)

Fig. S7. a) Fluorescence spectra of Alexa 488 as a function of the concentration of Trytophan (Trp) in 100 mM phosphate buffer. b) Stern-Volmer plot of Alexa 488 quenched by Trp. ([Alexa 488] = 0.42 M).

Fluorescence quenching mechanism

The fluorescence lifetime of Cytc-A488 is significantly shorter in the absence of GdHCl than

in its presence whereas the fluorescence lifetime of Alexa 488 is not affected by the addition of

GdHCl. From the fs-transient absorption measurement and the fluorescence quenching of Alexa 488

by the addition of Trp, we found that PET take place dominantly between the attached Alexa 488 and

amino acids such as Trp, and PET in the folded state of a protein is very efficient compared to that in

the denatured state (See the section of the transient absorption measurement and fluorescence

quenching of Alexa 488 by the addition of Trp). Indeed, Chen et al. reported that the fluorescence of

Alexa 488 is quenched by the interaction with Trp, Tyr, Met and His residues through a combination

of static and dynamic quenching mechanism.2 They also show that electron transfer from Trp to Alexa

488 is highly favorable compared with other three residues. These results agree well with our results.

Thus, we suggest that the fluorescence quenching of Cytc-A488 by GdHCl is mainly due to PET from

the electron donor (Trp) to Alexa 488.

Here, it is worthy to note that the fluorescence lifetime (3.27 ns) of the unfolded Cytc-A488

is slightly shorter than that observed from free Alexa 488 (3.69 ns), indicating that there is another

source for the fluorescence quenching of Alexa 488 in addition to PET between Alexa 488 and Trp.

As suggested by Chen et al., the fluorescence quenching of Cytc-A488 in the high concentration of

GdHCl may be due to the interaction with Tyr, Met and His residues. Especially, Tyr 97 and Cys102

are located to the same helix and the distance between two residues is short as depicted in Fig. S8 (The

distance between Tyr97 and cys102 is calculated to be 7.3Å from the crystal structure). Thus we

suggest that the interaction between Alexa 488 and Tyr97 should induce fluorescence quenching even

in the unfolded state as well as in the native state.


Fig. S8. Structure of Alexa 488-labeled yeast iso-1-cytochrome c (Cytc-A488) (Yeast iso-1-Cytochrome c: PDB ID code 1YCC) showing the heme and the locations of residues. Alexa 488 is labeled to residue 102 (Cys102) of Cytc. The distance between Cys102 (Orange) and Tyr97 (Pink) is calculated to be 7.3 Å from the crystal structure.

Fluorescence quenching of ApoCytc-A488 by the addition of GdHCl

500 520 540 560 580 600 620 6400.0

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

3.0x106

Flu

ore

sce

nce

Inte

nsi

ty

Wavelength / nm

ApoCytc in 0.0M GdHCl ApoCytc in 0.5M GdHCl ApoCytc in 1.0M GdHCl ApoCytc in 2.0M GdHCl ApoCytc in 4.0M GdHCl

4 6 8 10 12 14 16 18 20 22 241

10

100

Inte

nsi

ty

t / ns

Apo-Cytc in 0M GdHCl Apo-Cytc in 4M GdHCl

1=1.06 ns (0.32)2=3.41 ns (0.68)

1=1.09 ns (0.12)2=3.66 ns (0.88)

Fig. S9. a) Fluorescence spectra of ApoCytc-A488 as a function of the concentration of GdHCl in 100 mM phosphate buffer. b) Fluorescence decay profiles of ApoCytc-A488. The fluorescence lifetimes () of Alexa 488 attached to ApoCytc at 0 M and 4 M GdHCl were determined to be 2.66 ns and 3.35 ns, respectively.

Three-state model

In the three-state model, the native Cytc (N) is unfolded through an intermediate (I).

N I Um1 1 m2 2c ,m c ,m

At each concentration of the denaturant, the free energies for NI and IU transitions, G1 and G2,

were calculated using the following equation:

1

2

N i

i I

I i

i U

x xG RTln

x x

x xG RTln

x x

where xi is the numerical value of the structure-sensitive parameter at the ith denaturant concentration

(i.e., the fluorescence intensity in this case); xN, xI and xU are the numerical values of the same


parameter relative to the native, intermediate and completely unfolded states, respectively. The free

energy changes for NI and IU transitions in the absence of denaturant ( 0N-IG and 0

I-UG ) and

the -m1 and -m2 values were calculated from the plot of GObsd as a function of [GdHCl] with an

equation

GdHCl

GdHCl

0Obsd N-I

0Obsd I-U

G G m

G G m

Changes in -m values are indicators of the change in solvent-exposed surface area upon each transition.

Single-molecule experiments with confocal laser scanning microscopy

In order to take the single-molecule image, solutions of Cytc-A488 at 1 nM contained 1wt.%

trehalose. Trehalose (-D-glucopyranosyl--D-glucopyranoside) is well known as a stabilizer in

single-molecule experiments. However, it also has a stabilizing effect on proteins that is opposite to

the denaturing effect of GdHCl. Therefore, in this study trehalose was added to the sample solution at

less than 1% (25 mM)as a minimum quantity to minimize its stabilizing effect on proteins that is

opposite to the denaturing effect of GdHCl. As a control experiment, we measured the fluorescence

lifetimes of Cytc-A488 and Alexa 488 with 1% trehelose in the bulk phase. As shown in Fig. S10a, the

photophysical properties of Cytc-A488 and Alexa 488 are not affected by the addition of 1wt.%

Trehalose. A thin film was prepared by spin casting 40 L of the Cytc-A488 solution onto a

microscope cover slip and was dried in air. Denatured Cytc-A488 in a 1% trehalose film was prepared

by spin casting with Cytc-A488 solutions in a 100 mM phosphate buffer (pH 7.0) containing GdHCl at

various concentrations.

On the other hand, we have measured the fluorescence lifetimes of single-Cytc-A488 and

Alexa 488 in the absence of a denaturant. The fluorescence lifetimes of the folded Cytc-A488 and

Alexa 488 embedded in the trehalose film were determined to be 0.37 ns and 3.72 ns, respectively

(See Fig. S10b). These lifetimes are similar to those of Cytc-A488 and Alexa 488 measured in the

bulk experiment within the experimental error.

2 4 6 8 10 12 14 16 18 20 22 241

10

100

1000

Cytc-A488 without Trehalose Cytc-A488 with 1% Trehalose Alexa 488 without Trehalose Alexa 488 with 1% Trehalose

Fl.

Inte

nsi

ty /

a.u

.

4 6 8 10 12 14 16 18 20 22 24

0

20

40

60

80

100 Cytc-A488 Alexa 488

t / ns t / ns

a) b)

Fig. S10. a) Trehalose effect on the fluorescence lifetimes of Cytc-A488 and Alexa 488 with and without GdHCl in 100 mM phosphate buffer solutions. b) Fluorescence decay profiles of Cytc-A488 (black) and Alexa 488 (blue) embedded in the trehalose film (Ex = 485 nm). The theoretical fits obtained from the fitting analysis are shown in red.


Fluorescence intensity trajectories of Cytc-A488 and Alexa 488

Fig. S11 exhibits typical fluorescence intensity trajectories (FITs) of single-molecule Cytc-

A488 with various GdHCl solutions in 100 mM phosphate buffer (pH 7.0). As shown in Fig. S11, the

FITs of Cytc-A488 with various GdHCl solutions exhibit one-step photo-bleaching and on-off

blinking behaviors, which are characteristic features of a single-molecule. Especially, FITs of Cytc-

A488 in the trehalose film containing the small amount of GdHCl (≤1.0M) shows the strong

fluctuation compared with that of single-molecule Alexa 488 on a glass coverslip, indicating that PET

in the same protein take place substantially between Alexa 488 and amino acids such as Trp, and the

PET in the folded state of a protein is much more efficient than in the denatured state.

Fig. S11. Typical fluorescence trajectories of single-molecule Alexa 488 (a) in buffer solutions without a denaturant and Cytc-A488 (b-f) with various concentrations of GdHCl. b-f) [GdHCl]= 0.0, 0.5, 1.0, 2.0 and 4.0 M.

Fluorescence Correlation Spectroscopy (FCS)

In order to elucidate the contribution of the singlet-triplet relaxation of Alexa 488 in the FCS

curve of Cytc-A488, we observed the FCS curve for Alexa 488 at various concentrations of GdHCl at

room temperature. However, we could not observe any dynamics due to the singlet-triplet relaxation

as depicted in Fig. S12a. Furthermore, in FCS experiment free Alexa 488 did not show fast dynamics

(< 0.01 ms) in the presence of BSA and Tween-20 (See Fig. S12b). Thus, we did not consider the

contribution ofthe singlet-triplet relaxation of Alexa 488. Mukhopadhyay et al. also reported that in the

FCS experiment, no fast component (< 0.01 ms) was observed for free Alexa 488 in buffer.8

0 5 10 15 20 25 30

0 5 10 15 20 25 30 35 40

time / s0 5 10 15 20 25 30 35 40

time / s

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25

0 5 10 15 20 25 30

0 1 2 3 4 5 6 7 8 9 10

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

time / s

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

time / s

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25 30 35 40

0 5 10 15 20 25 30 35 40

time / s

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

time / s0 1 2 3 4 5 6 7 8

a) b) c)

d) e) f)


Fig. S12. a) FCS data recorded from Alexa 488 at various concentrations of GdHCl at room

temperature. b) FCS data recorded from Alexa 488 with BSA and Tween-20 at various concentrations

of GdHCl at room temperature.

References

(1) Mei, E.; Tang, J.; Vanderkooi, J. M.; Hochstrasser, R. M. J. Am. Chem. Soc. 2003, 125, 2730-

5.

(2) Chen, H.; Ahsan, S. S.; Santiago-Berrios, M. B.; Abruna, H. D.; Webb, W. W. J. Am. Chem.

Soc. 2010, 132, 7344-45.

(3) Neuweiler, H.; Johnson, C. M.; Fersht, A. R. Proc. Natl. Acad. Sci. U S A. 2009, 106, 18569-

74.

(4) Widengren, J.; Mets, U.; Rigler, R. J. Phys. Chem. B 1995, 99, 13368-13379.

(5) Bonnet, G.; Krichevsky, O.; Libchaber, A. Proc. Natl. Acad. Sci. U S A 1998, 95, 8602-6.

(6) Levene, M. J.; Korlach, J.; Turner, S. W.; Foquet, M.; Craighead, H. G.; Webb, W. W. Science

2003, 299, 682-6.

(7) Ries, J.; Yu, S. R.; Burkhardt, M.; Brand, M.; Schwille, P. Nat. Methods 2009, 6, 643-5.

(8) Mukhopadhyay, J.; Kapanidis, A. N.; Mekler, V.; Kortkhonjia, E.; Leroy, O.; Ebright, Y. W.;

Weiss, S.; Ebright, R. H. Biophys. J. 2002, 82, 115a-116a.

1E-4 1E-3 0.01 0.1 1 10 100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

G(

) No

rmal

ize

d

t / ms

Alexa 488 in 0M GdHCl Alexa 488 in 4M GdHCl

1E-4 1E-3 0.01 0.1 1 10 100

0.0

0.5

1.0

1.5

2.0 Alexa 488 in 0M GdHCl with BSA and Tween-20 Alexa 488 in 4M GdHCl with BSA and Tween-20

G(

) No

rma

lize

d

t / ms

a) b)


unfolding dynamics of cytochrome c revealed by single … · 2011-02-07 · unfolding dynamics of...

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