Supplementary information
Tryptophan-Tryptophan Energy Migration as a Tool to Follow ApoflavodoxinFolding
Nina V. Visser, Adrie H. Westphal, Arie van Hoek, Carlo P. M. van Mierlo, Antonie J. W. G. Visser, Herbert van Amerongen
Provided as Supplementary Material are: an example of the analysis of fluorescence decay and fluorescence anisotropy decay data of the apoflavodoxinvariant WFF (SI1), the fluorescence emission spectra of WT apoflavodoxin and variants of apoflavodoxins (SI2), the simulation of the fluorescence anisotropy decay of WT apoflavodoxin (SI3), the determination of steady-state fluorescence anisotropy data derived from polarized time-resolved fluorescence data (SI4), the spectral overlap integral and centre of gravity of the fluorescence band of WT apoflavodoxin both as a function of GuHCl concentration, (SI5), and finally, the analysis of the GuHCl-dependent fluorescence anisotropy decay data of the WFW apoflavodoxin variant (SI6).
Supplementary material: 1
Example of the analysis of total fluorescence decay and fluorescence anisotropy decay of apoflavodoxin
Total fluorescence decay analysis
The fluorescence lifetime profile that consists of a sum of discrete exponentials with lifetimesi and amplitudesi can be retrieved from the total fluorescence I(t) (I(t) = I||(t) + 2 I(t)) through:
S1-1
E(t) is the instrumental response function and denotes a convolution product.
In Fig. S1-1 the total fluorescence decay data (experimental and fitted) of WFF apoflavodoxin are presented. To obtain an optimal fit to the experimental data a sum of three exponential terms (N=3) is required (see legend to figure).
Figure S1-1. Total fluorescence decay (grey) and associated fit (black) obtained for 4 M WFF apoflavodoxin in 100 mM potassium pyrophosphate, pH 6.0, 25 0C. The excitation wavelength is 300 nm and the emission is measured at 348.8 nm. The optimized lifetimes and relative amplitudes (percentage in parentheses) obtained are: 1=0.11±0.01 ns (11.9%), 2=1.92±0.04 ns (27.8%) and 3=4.30±0.02 ns (60.3%), the goodness-of-fit parameter χ2 = 1.00 (when 2 is minimal, i.e., 2 = 1, an optimal fit is obtained). To illustrate the quality of the fit the weighted residuals and autocorrelation function of the residuals are shown as well.
Fluorescence anisotropy decay analysis
In case of fluorescence anisotropy decay analysisthe time-dependentfluorescence anisotropy r(t)(r(t) = (I||(t) – I(t))/ I(t)) is calculated using the parallel I||(t)and perpendicular I(t) fluorescence intensity components and the following links:
S1-2
S1-3
We assume that all fluorescence lifetimes i contributeequally to apoflavodoxin’s anisotropy decay that is described by a summation of exponential functions with correlation times j and amplitudes r0j as adjustable parameters. In eqs. S1-2 and S1-3 i≠ j, i.e., the uncorrelated or non-associative model is used. After -pulse excitation the time-dependent anisotropyr(t) is described by:
S1-4
After optimizing the fit of the total fluorescence decay (see above), parameters i and i are fixed during the analysis of the fluorescence anisotropy decay. The individual intensities I||(t) and I(t) are globally analyzed using equations S1-1 to S1-4. In case of WFF apoflavodoxin eq.S1-4 with M=1 suffices to obtain an optimal fit of the anisotropy decay data (see Fig. S1-2).
Figure S1-2. Fluorescence anisotropy decay (grey) and associated fit (black) obtained for 4 M WFF apoflavodoxin in 100 mM potassium pyrophosphate, pH 6.0, 25 0C. The excitation wavelength is 300 nm and the emission is measured at 348.8 nm. The optimized parameters are: correlation time1 = 10.4±0.1 ns and amplitude r01 = 0.238; the goodness-of-fit parameter χ2 = 1.04 (when 2 is minimal, i.e., 2 = 1, an optimal fit is obtained). To illustrate the quality of the fit the weighted residuals and autocorrelation function of the residuals are shown as well.
Supplementary material: 2
Fluorescence spectra of WT and variants of apoflavodoxin
Normalizedsteady-state fluorescence spectra of native WT apoFDand apoFD variants with one or two tryptophans are shown in Fig. S2-1.
Figure S2-1.Normalizedsteady-state fluorescence spectra of native WT apoflavodoxin (blue) and apoflavodoxin variants WFF (red), WFW (green), WWF (yellow). All apoflavodoxin concentrations are 4 M in 100 mM potassium pyrophosphate, pH 6.0, 25 C. The excitation wavelength is 300 nm and excitation and emission slit widths are 2 nm.
Supplementary material:3
Simulation of fluorescence anisotropy decay of WT apoflavodoxin
In Fig. S3-1A the relative orientations of the three tryptophan residues of native WT apoflavodoxin are shown together with the possible pathways of resonance energy transfer. The rate constants for resonance energy transfer between two tryptophans (i.e., kT) are calculated using eq. 3 (see main text). The overlap integrals J are calculated using the absorption and emission spectra of the tryptophan residues in WT apoflavodoxin.Both 2 values and distances R are retrieved from the 3D-structure of flavodoxin (Alagaratnamet al., 2005) and are collected in Table S3-1. Correlation times for unidirectional resonance energy transfer are calculated using eq. 4 (see main text).
All tryptophans of apoflavodoxin are assumed to become excited with equal probability. Consequently, theanisotropy decay of native apoflavodoxin is the result of 3 processes: i) excitation of W128 leads to depolarization that is only due to overall protein rotation (r=10.4 ns); ii) excitation of W167 leads to rapid depolarization due to energy transfer to W128 with rate constant kT1 = 34.7 ns-1 (T1 = 0.03 ns) and the corresponding amplitude (T1) depends on the mutual orientation of W167 and W128; iii) excitation of W74 leads to slow transfer to W167 with rate constant kT2= 0.14 ns-1(T2 = 7.2 ns), which is followed “instantaneously” by transfer to W128, and the corresponding amplitude (T2) in the anisotropy decay depends on the relative orientations of W74 and W128. Amplitudes are calculated using the following equations:
S3-1
S3-2
T is the angle between donor and acceptor dipole moments.The brackets < > denote the average of two directions, i.e., donor1→acceptor2 and donor2→acceptor1. The anisotropy decay of each individual tryptophan residue (ri(t)) can be simulated because it is described by the following equation:
ri(t)={T1exp(-t/T1)+ r}exp(-t/r)S3-3
By using the relationship the overall anisotropy of apoflavodoxin can be calculated. The results, which are scaled to the value of the measured initial anisotropy r(0)=0.16, are presented in Fig. S3-1B-C. The simulated anisotropy decay of native apoflavodoxin, which is calculated by averaging the three simulated fluorescence anisotropy decays of the individual tryptophan residues of apoflavodoxin, is in fair agreement with the fit of the experimentally obtained anisotropy decay data of native apoflavodoxin (Fig. 2B, see main text). Thus, the discussed energy transfer pathways between tryptophans adequately describe the experimental fluorescence anisotropy decay of native apoflavodoxin.
Figure S3-1. A: Schematic view of the mutual orientations of the three tryptophans of native WT flavodoxin from A. vinelandii. Solid and dashed arrowsshow the possible pathways of energy transfer. Arrows in the molecular plane of the individual tryptophan residues show the transition dipole moments. B: Simulated fluorescence anisotropy decays of each excited tryptophan residue: curve 1 – W128; curve 2 – W74; curve 3 - W167. C: Fit of the model equation described in the legend of Fig. 2 (see main text) to the experimental fluorescence anisotropy decay data of native WT apoflavodoxin in 100 mM potassium pyrophosphate pH 6, 25 0C (dashed line). In addition, the average of the three simulated fluorescence anisotropy decays of the individual tryptophan residues of apoflavodoxin (see B) is shown (solid line).
Table S3-1. Energy transfer parameters obtained for the three tryptophan pairs of A. vinelandii flavodoxin as calculated using the corresponding X-ray structure (pdb code 1YOB)
W → W / R(Å) / R0
(Å) / 2 / kT
(ns-1) / T
(degrees)
167 → 128 / 6.8 / 14.2 / 0.87 / 34.7 (kT3) / 87
74 → 128 / 19.5 / 11.1 / 0.2 / 0.014 (kT2) / 60
74 → 167 / 15.0 / 12.6 / 0.44 / 0.14 (kT1) / 107
R is the distance between donor and acceptor (i.e., the distance between particular tryptophans); R0 is the Förster distance, 2 is the orientation factor, kT is the rate constant for resonance energy transfer between a pair of tryptophan residues, and T is the angle between donor and acceptor dipole moments.
Supplementary material:4
Steady-state fluorescence anisotropy obtained from polarized time-resolved fluorescence
The availability of time-resolved fluorescence data of apoflavodoxin enables the calculation of the corresponding steady-state fluorescence anisotropy values <r> by using the following relation (Lakowicz, 2006):
S4-1
I(t) is the total fluorescence decay (I(t) = I||(t) + 2 I(t)) and r(t) is the time-dependent fluorescence anisotropy (r(t) = (I||(t) – I(t))/ I(t)). The excitation wavelength is 300 nm and the fluorescence is detected at 348.8 nm.
Steady-state fluorescence anisotropy values reconstructed from time-resolved data of WT apoFD and apoFD variants as a function of denaturant (GuHCl) concentration are plotted in Fig. S4-1. The directly measured steady-state fluorescence anisotropy values of WT apoFD (Bollenet al., 2004) are incorporated to illustrate the agreement between both data sets.
Figure S4-1.Steady-state fluorescence anisotropy of WT apoflavodoxin and apoflavodoxin variants as a function of concentration denaturant (GuHCl). Directly measured steady-state anisotropy values of wild type apoflavodoxin (open black circles) are taken from (Bollen 2004). Reconstructed steady-state anisotropy values, applying eq. S4-1 on time-resolved data obtained in this study are shown as closedcoloured circles: WT apoflavodoxin (blue)and apoflavodoxin variants WFF (red), WFW (green), WWF (yellow).The excitation wavelength is 300 nm and emission is measured at 348.8 nm. Apoflavodoxin anisotropy is measured at 25 0C in 100 mM potassium pyrophosphate, pH 6.0.
Equation S4-1 can be rewritten into the well known Perrin equation (Lakowicz, 2006), if we assume that both the time-dependent fluorescence intensity I(t) and the anisotropy r(t) follow an exponential decay law:
S4-2
S4-3
> is the average fluorescence lifetime and <> is a harmonic mean correlation time characteristic for the particular fluorescence depolarization process. After substituting equations S4-2 and S4-3 into eq. S4-1 and carrying out the integrations one obtains the Perrin equation:
S4-4
Since the steady-state fluorescence anisotropy depends on the average fluorescence lifetime, we have plotted (for completeness) the measured average fluorescence lifetimes of WT and all variants of apoflavodoxin used as a function of concentration GuHCl in Fig. S4-2.
Figure S4-2. Dependence of average fluorescence lifetime of WT apoflavodoxin and variants ofapoflavodoxin (blue: WT; red: WFF;green: WFW;yellow: WWF) on concentration GuHCl. The data are obtained from analysis of the time-resolved fluorescence intensity data (see SI1) and are presented as the amplitude-averaged fluorescence lifetimes.The excitation wavelength is 300 nm and emission is measured at 348.8 nm. Apoflavodoxin is in 100 mM potassium pyrophosphate, pH 6.0, 25 0C.
Supplementary material:5
Spectral overlap integral and center of gravity of the fluorescence band of WT apoflavodoxin as a function of GuHCl concentration
The rate constant of resonance energy transfer kTis given by the Förster rate equation:
S5-1
is the orientation factor for the relevant transition dipole moments, n the refractive index of the donor-acceptor intervening medium, krthe radiative rate constant (ns-1), J the integrated spectral overlap of the (acceptor) tryptophan absorbance and (donor) fluorescence spectra (M-1cm3), and R the distance (nm) between the donor and acceptor. The transfer rate constant can be recasted into the following form:
S5-2
Dis the donor fluorescence lifetime in the absenceof acceptorand R0 the critical transfer distance (or Förster distance)at which the rate of transfer is equal to the fluorescencedecay rate:
S5-3
QD is the quantum yield of donor fluorescence in absence of acceptor. The overlap integral between the fluorescence spectrum of the donor and the molar absorption spectrum of the acceptor is given by:
S5-4
FD()is the fluorescence spectrum of the donor, normalized such that the integrated spectrum is equal to unity, and A()is the absorption spectrum of the acceptor.The critical transfer distance R0is in units of Ångstrom, whereas is in nanometers, Ain M-1cm-1and J in nm4M-1cm-1. The center of gravity of the emission cg is calculated using:
S5-5
I() is the fluorescence intensity at wavelength and the summation is done in this case from 310 nm to 450 nm. The spectral overlap integrals are determined using molar absorption spectra and corrected fluorescence spectra taken at 1-nm increments of 4 M WT apoflavodoxin at increasing concentrations GuHCl. The centers of gravity are calculated using the fluorescence spectra mentioned. Both J and cg are plotted as a function of GuHCl concentration in Fig. S5-1 and are correlated: a decrease of J is accompanied by an increase of cg. This correlation is in agreement with the observation that the absorption spectra of apoflavodoxin are hardly dependent on the presence of GuHCl, whereas the emission spectra show a progressive red shift upon increasing the concentration GuHCl.
Figure S5-1. Dependence of the spectral overlap integral J(●) and the center of gravity of fluorescence emission cg(○) of WT apoflavodoxin on GuHCl concentration. Apoflavodoxin is in 100 mM potassium pyrophosphate, pH 6.0, 25 0C.
It is illustrative to investigate the effect of varying the non-geometric factors in eqs. S5-1 and S5-3 on the values of the critical transfer distance R0 and the actual distance R. An empirical relationship between the radiative rate constant kr and the wavelength of maximum intensity of the tryptophan fluorescence spectrum has been previously derived (Engelborghs, 2003) and is plotted for apoflavodoxin as a function of the concentrationGuHCl in Fig. S5-2.
Figure S5-2. Dependence of the radiative rate constant kr(●) of WT apoflavodoxin on GuHCl concentration. Apoflavodoxin is in 100 mM potassium pyrophosphate, pH 6.0, 25 0C.
Fig. S5-2 shows that kr changes from 0.052 ns-1 (native apoflavodoxin) to 0.043 ns-1 (apoflavodoxin in 4 M GuHCl). In addition, eq. S5-1 shows that rate constant kT is proportional to both kr and R-6. Under the assumption that the other variables in eq. S5-1 remain constant, a reduction of the transfer rate constant kT by a factor of 1.21 (i.e., 0.052/0.043) causes an increase in distance (R) of only 3%. Equation S5-3 shows that R0 depends on factors like spectral overlap integral J and refractive index n. Toptygin et al. (Toptyginet al., 2002) have shown that tryptophans in a native protein environment sense a relatively high refractive index value of 1.6. A tryptophan in an unfolded protein at 4M GuHCl senses the refractive index of the denaturant solution, i.e., n 1.4. Under the assumption that the other variables in eq. S5-3 remain constant, a reduction in n from 1.6 to 1.4 results in a 9% increase in R0. Similarly, a decrease of J by a factor of 2.8 leads to a 15% decrease in R0, when all othervariables in eq. S5-3 remain constant. Note that n and J have antagonistic effects on the value of R0. The general conclusion from the inspection of the effects the discussed parameters have on kT is that geometric factors (i.e., R and 2) are the main determinants of the magnitude of the transfer rate constant kT.
Supplementary material:6
Analysis of fluorescence anisotropy decay data of WFW apoflavodoxin obtained as a function of GuHCl concentration
It is interesting to use time-dependent fluorescence anisotropy to monitor the onset of flexibility of tryptophans upon GuHCl-induced unfolding of apoflavodoxin. Here, we present a detailed analysis of the fluorescence anisotropy decay of the WFW apoflavodoxin variant as a function of [GuHCl]. In this apoflavodoxin variant FRET can only occur between W74 and W167 and thus information is obtained about two depolarization sources, namely FRET and protein rotation. Indeed, without GuHCl only two correlation times (3.3 ns → FRET; 10.4 ns → protein rotation) are required to obtain an optimal fit of the data (see Table S6-1: 1, 1, r, r). These two correlation times are sufficient to describe the fluorescence anisotropy decay up to 1.7 M GuHCl. At higher concentrations GuHCl an extra correlation time of 0.2-0.3 ns is needed to obtain an optimal fit to the data and its amplitude contribution increases as the concentration GuHCl becomes higher (see Table S6-1: 2, 2). The short correlation time of 0.2-0.3 ns describes rapid internal motion of tryptophan residues (‘rattling in cage’), whereas the corresponding amplitude reflects the angular freedom of the tryptophans. Characteristic observations are: i) the standard errors show that the 2-values do not interfere with the 1-values throughout the whole range of GuHCl concentrations used, in other words: the correlation times have distinct values and no cross-talk occurs; ii) the anisotropy amplitude associated with the correlation time that describes FRET between W74 and W167 has a value of about 0.1. Up to 1.7 M GuHCl, the remaining anisotropy amplitude value is associated with protein rotation. Above 1.7 M GuHCl, the latter amplitude is distributed between overall protein rotation (r) and fast internal motions of tryptophans (2). These features show that FRET between W74 and W167 is independent of the motional processes observed. In fig. S6-1 a few fitted anisotropy decay curves of apoflavodoxin are shown for illustration purposes.
Figure S6-1. Fitted fluorescence anisotropy decay curves of apoflavodoxin variant WFWin 100 mM potassium pyrophosphate, pH 6.0, 25 0C. Black: no GuHCl; red: 1.8 M GuHCl; blue: 2.1 M GuHCl; green: 3.0 M GuHCl. The excitation wavelength is 300 nm and the emission is measured at 348.8 nm. All fitting parameters are summarized in Table S6-1.
Table S6-1. Optimized parameters that describe the fluorescence anisotropy decay of WFW apoflavodoxin as a function of GuHCl concentration.
GuHCl (M) / 1(-) / 1
(ns) / 2
(-) / 2
(ns) / r
(-) / r, fix
(ns) / 2
0 / 0.10
(0.08-0.12) / 3.3
(3.0-3.6) / - / - / 0.15
(0.12-0.18) / 10.4 / 1.02
0.5 / 0.08
(0.06-0.09) / 2.8
(2.4-3.2) / - / - / 0.17
(0.14-0.20) / 10.4 / 1.01
0.9 / 0.05
(0.04-0.06) / 1.9
(1.7-2.1) / - / - / 0.19
(0.15-0.23) / 17 / 1.10
1.8 / 0.06
(0.05-0.07) / 1.5
(1.3-1.7) / 0.03
(0.03-0.03) / 0.22
(0.18-0.26) / 0.13
(0.10-0.16) / 20 / 1.05
2.1 / 0.10
(0.08-0.12) / 1.5
(1.3-1.7) / 0.03
(0.03-0.03) / 0.32
(0.26-0.38) / 0.12
(0.10-0.14) / 20 / 1.06
2.6 / 0.12
(0.10-0.14) / 1.6
(1.4-1.8) / 0.07
(0.06-0.08) / 0.27
(0.22-0.32) / 0.05
(0.04-0.06) / 25 / 1.04
3.0 / 0.11
(0.09-0.13) / 1.5
(1.3-1.7) / 0.08
(0.07-0.09) / 0.33
(0.26-0.40) / 0.03
(0.02-0.04) / 25 / 1.05
For explanation of parameters see text. Values in parentheses are the standard errors obtained from the fit. The rotational correlation time of the protein (r) has been fixed during the analysis. The value of 2 denotes the quality of the fit (when 2 is minimal, i.e., 2 = 1, an optimal fit is obtained).
Supplementary material: References
Alagaratnam, S., G. van Pouderoyen, T. Pijning, B. W. Dijkstra, D. Cavazzini, G. L. Rossi, W. M. A. M. Van Dongen, C. P. M. van Mierlo, W. J. H. van Berkel, and G. W. Canters. 2005. A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential. Protein Science 14:2284-2295.
Bollen, Y. J. M., I. E. Sánchez, and C. P. M. van Mierlo. 2004. Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin. Biochemistry 43:10475-10489.
Engelborghs, Y. 2003. Correlating protein structure and protein fluorescence. Journal of Fluorescence 13:9-16.
Lakowicz, J. R. 2006. Principles of fluorescence spectroscopy. New York, USA: Springer Science+Bussiness Media, LLC.
Toptygin, D., R. S. Savtchenko, N. D. Meadow, S. Roseman, and L. Brand. 2002. Effect of the solvent refractive index on the excited-state lifetime of a single tryptophan residue in a protein. J. Phys. Chem. B 106:3724-3734.