Online Resource, Submitted to Photosynthesis Research.
Reconstituted CP29: Multicomponent
fluorescence decay from
an optically
homogeneous sample.
Erica Belgio*, Giorgio Tumino*, Stefano Santabarbara, Giuseppe Zucchelli and Robert
Jennings+
Consiglio Nazionale delle Ricerche, Istituto di Biofisica, sede di Milano and
Dipartimento di Biologia, Università degli Studi di Milano, via G. Celoria 26, 20133
Milano, Italy
+
Corresponding
author:
robert.jennings@unimi.it;
tel.
+390250314858;
Fax:
+390250314815
*Equal contribution
1
Determination of the sensitivity of the absorption-fluorescence analysis in terms of the
Stepanov relation in revealing spectral heterogeneity of Chl-protein complexes.
In this paper we have critically examined the suggestion that the multiexponential decay of fluorescence detected in Chlorophyll-binding complexes, CP29 in
this case, is associate with different conformers of the antenna complex (e.g. Crimi et al.
2001; Avenson et al. 2007; Johnson et al. 2009; Moya et al. 2001, Pascal et al. 2005,
van Oort et al. 2007). As the chlorophyll absorption/fluorescence bands are sensitive to
distortions of the tetrapyrrole ring which are brought about by the conformation of the
binding sites themselves (Zucchelli et al. 2007), one would expect that the presence of
different conformers would lead to spectral heterogeneity. Such heterogeneity is
expected to lead to a substantial deviation between the measured and the calculated
emission spectra (van Metter and Knox, 1976) using the thermodynamic
absorption/fluorescence relation elaborated by Stepanov (1956; Equation 1.S)
F() A()  D(T) e
2

h
k BT
(1.S)
which implies complete thermalisation of the excited state. Although derived for a
pigment solution the Stepanov equation describes satisfactorily the emission of coupled
pigment systems, such as chlorophyll protein complexes (e.g. van Metter and Knox
1976; Zucchelli et al. 1995; Jennings et al. 2000; Jennings et al. 2003; Dau 1996;
Tumino et al. 2008, Giuffra et al. 1997; Belgio et al. 2010). It provides accurate
information in the wavelength region where absorption and fluorescence overlap, which
for CP29 is between about 660 – 690 nm.
2
The important question is the spectral resolution of the approach in detecting
absorption/fluorescence heterogeneity in this CP29 sample for the situation described in
the fluorescence decay measurements, in which the relative amplitudes of the two
principle decay components are 0.6 and 0.4 and the relative fluorescence yields of these
two components are both 0.5. Both parameters will contribute to the simulation of
spectroscopic heterogeneity.
To this end we have simulated emission spectra for a number of conditions in
which sample absorption heterogeneity was assumed. In the first case the CP29
absorption spectrum was translated by various small wavelength intervals, of up to 1
nm, both towards longer and shorter wavelengths. When the two wavelength-shifted
absorption spectra were summed, using the weighting factors of 0.4 and 0.6, derived
from the fluorescence decay amplitudes (Figure 1 of the main text), the resulting
composite spectrum is similar to the measured CP29 absorption spectrum (data not
presented). The fluorescence spectra were then calculated (equation 1.S): (i) for the
summed absorption spectra, using the 0.4 and 0.6 weighting factors based on the
fluorescence decay amplitudes; ii) for each of the wavelength shifted absorption spectra
separately and which were subsequently summed using the 0.5 and 0.5 weighting
factors derived from the fluorescence yields of the 3 ns and 5 ns decay components. The
situation (i) simulates the case in which the sample is assumed homogeneous and
thermally equilibrated. The situation (ii) simulates the case in which the sample contains
two different, uncorrelated, spectroscopic subpopulations. The calculated emission
spectra for the cases (i) and (ii) and for three wavelength shifts (0.25, 0.5, 1.0 nm) are
shown in Figure S1 for the 10 nm interval around the wavelength maximum. For the 1
nm shift the thermally equilibrated and homogeneous case (i) is clearly red shifted with
3
respect to the two different subpopulations case (ii) and their fluorescence difference
spectra underline this (Figure S2). For the 0.5 nm shift the fluorescence red shift is just
discernable, but it is clear that we are on the resolution borderline (Figure S1). Instead
for the 0.25 nm shift the spectral shifting which is generated is well below resolution.
In the above described simulations the heterogeneity examined was exclusively a
wavelength heterogeneity, maintaining constant the absorption band shapes. As this is
probably not realistic, we have performed similar calculations in which the band shapes
were also modified either to the long or the short wavelength side. An example of this is
also shown in Figures S1 and S2 for the case of a 1 nm spectral shift together with a
10% broadening on the long wavelength side. This shows that spectral broadening has
the effect of enhancing the difference between (i) and (ii).
It is evident from these simulations of sample heterogeneity that the Stepanov
approach which we have used has quite a high resolution and can probably detect
sample heterogeneity differences down to about 0.5 nm in terms of the wavelength
position of CP29 absorption, with or without spectral broadening or narrowing. The
experimental data (Figure 5 of the main text) do not indicate such differences and so we
conclude that if sample absorption heterogeneity does exist it must be no greater than
0.5 nm for the recombinant CP29 sample.
4
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7
Figure S1. Simulated fluorescence emission spectra assuming absorption sample
heterogeneity. Panels A, B and C show the calculated emission in the 676 – 686 nm
interval for the cases in which the CP29 absorption spectra were translated along the
wavelength axis by +0.25 nm, +0.5 nm and +1 nm respectively with respect to the
measured spectrum. The wavelength-translated spectra are indicated by the dashed line
and the non translated spectrum by the continuous line. Panel D shows the effect of a
10% band broadening on the long wavelength side of the absorption spectrum together
with the 1 nm wavelength translation (Panel C).
8
Figure S2. Calculated difference spectra of the wavelength translated minus non
translated spectra reported in Figure 6. Continuous line: +0.25 nm shift; dashed line:
+0.5 nm shift; dashed-dotted line: +1 nm shift; dashed-dotted-dotted line: 1 nm shift
plus 10% band broadening".
9