1
Supplementary Materials
2
3
Thermal protein unfolding of HKR1 rhodopsin
4
The thermal stability of HKR1 in HEPES/DDM pH 7.4 buffer was studied by stepwise
5
sample heating and subsequent sample cooling in a thermostatic heat bath whereby
6
transmission spectra T() were measured (S1,S2). The light transmission is determined by
7
light absorption and light scattering according to
8
T ( )  Ta ( )Tsca ( )  exp[  ( )]  exp{ [ a ( )   sca ( )]} ,
9
where a is the absorption coefficient, sca is the scattering coefficient, and  the sample
10
(S1)
length.
11
The applied temporal heating profile is shown in the inset of Fig.S1. Attenuation
12
coefficient spectra belonging to some sample temperatures are shown in the main part of
13
Fig.S1. Up to about 45 °C the light attenuation was dominated by light absorption. Further
14
heating-up caused progressive increasing light scattering due to protein unfolding with
15
subsequent aggregation (S1). In the temperature step from 51.1 °C to 55.8 °C the attenuation
16
coefficient increased from (380 nm) = 3.4 cm-1 to (380 nm) = 8.4 cm-1 indicating vigorous
17
protein unfolding (protein melting). During cooling down the sample to room temperature, the
18
light scattering continued to increase slightly, showing that the protein unfolding was
19
irreversible (S1-S4).
20
The development of the attenuation coefficient  at pr = 380 nm during sample heating
21
and cooling is depicted in Fig.S2. The strong rise of light attenuation in the heating step from
22
51.1 °C to 55.8 °C indicates protein melting. The apparent HKR1 melting temperature (S1-
23
S4) for the applied heating profile is m = 53.5±1 °C.
1
2
Figure S1. Attenuation coefficient spectra of HKR1 rhodopsin at certain times and
3
temperatures along the heating – cooling cycle shown in the inset.
4
1
2
Figure S2. Attenuation coefficient development at 380 nm versus temperature for applied
3
heating – cooling cycle of inset in Fig.S1. Obtained apparent protein melting temperature m
4
is indicated.
5
6
7
8
1
Förster-type energy transfer in HKR1 rhodopsin
2
The occurrence of Förster-type energy transfer is rationalized by comparison of the critical
3
Förster distance R0 with the globular HKR1 rhodopsin protein radius rHKR1. Assuming
4
spherical shape the globular protein radius is given by
5
 3

rHKR1  
VHKR1 
 4

6
where VHKR1 is the protein molecule volume, MHKR1  34520 g mol-1 is the protein molar
7
mass, NA is the Avogadro constant, and pr  1.4 g cm-3 (S5) is the protein mass density.
8
Insertion of values in Eq.S2 gives rHKR1  2.1 nm. The critical Förster distance is given by
9
(S6,S7)
1/ 3
1/ 3
 3 M

HKR1 

 4 N A  pr 


,
1/ 6
(S2)
1/ 6
 9 2F ,D

 
E ( ) A ( )4 d 
5 4  F ,D
 128 n

10
 9 2

R0  
E ( ) A ( )4 d 
5 4  F ,D
 128 n

11
where  is orientation factor of the transition dipoles, n  1.33 is the average refractive index
12
of the solution in the overlap region of absorption and emission of acceptor and donor, EF,D()
13
is the fluorescence quantum distribution of the donor in the absence of the acceptor, and A()
14
is the absorption cross-section of the acceptor. EF , D ( ) is the normalized fluorescence
15
quantum distribution of the donor according to EF , D  EF , D / F , D , where F,D is the
16
fluorescence quantum yield of the donor in the absence of the acceptor.
,
(S3)
17
The absorption cross-section spectrum of Trp and the fluorescence quantum distribution
18
of Tyr in the spectral overlap region are shown in Fig.S3 (curves derived from (S8)). The
19
absorption cross-section spectra of RetA and RetB (from Fig.4) and the fluorescence quantum
20
distribution of Trp (taken from (S8)) are shown in Fig.S4. The calculated critical Förster
21
distances are R0(Trp/Tyr)  1.57 nm, R0(RetA/Trp)  3.64 nm and R0(RetB/Trp)  3.06 nm
1
whereby the statistical isotropic orientation factor of the transition dipole moments of  2 =
2
2/3 was used (S6,S7). The determined critical Förster distance R0(Trp/Tyr) is comparable to
3
the HKR1 protein radius (note that 12 Tyr and 10 Trp are in one HKR1 rhodpsin), and the
4
determined critical Förster distances R0(RetA/Trp) and R0(RetB/Trp) are larger than the
5
HKR1 rhodopsin radius. These parameters indicate efficient occurrence of Förster-type
6
energy transfer.
7
8
9
Fig.S3: Absorption cross-section spectrum of Trp (from (S8)) and fluorescence quantum
distribution of Tyr (from (S8)).
1
2
3
Fig.S4: Absorption cross-section spectrum of RetA (from Fig.4) and fluorescence quantum
distribution of Trp (from (S8)).
4
5
References:
6
S1. Penzkofer, A., M. Stierl, P. Hegemann and S. Kateriya (2011) Thermal protein unfolding
7
in photo-activated adenylate cyclase nano-clusters from the amoeboflagelate Naegleria
8
gruberi NEG-M strain. J. Photochem. Photobiol. A: Chem. 225, 42-51.
1
S2. Penzkofer, A., M. Tanwar, S. K. Veetil, S. Kateriya, M. Stierl and P. Hegemann (2013)
2
Photo-dynamics and thermal behavior of the BLUF domain containing adenylate cyclase
3
NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain. Chem.Phys. 412,
4
96-108.
5
S3. Lepock, J. R., K. P. Ritchie, M. C. Kolios, A. M. Rodahl, K. A. Heinz and J. Kruuv
6
(1992) Influence of transition rates and scan rate on kinetic simulations of differential
7
scanning calorimetry profiles of reversible and irreversible protein denaturation. Biochem.
8
31, 12706-12712.
9
10
11
12
13
14
15
16
17
18
S4. Fujita, S. C., N. Gö and K. Imahori (1979) Melting-profile analysis of thermal stability of
thermolysin. A formulation of temperature-scanning kinetics. Biochem. 18, 24-28.
S5. Fischer, H., I. Polikarpov and A. Craievich (2004) Average protein density is a molecularweight-dependent function. Prot. Sci. 13, 2825-2828.
S6. Förster, Th. (1951) Fluoreszenz organischer Verbindungen. Vandenhoeck und Ruprecht,
Göttingen, Germany.
S7. Fleming, G. R. (1986) Chemical Applications of Ultrafast Spectroscopy. Oxford
University Press, New York.
S8. Lindsey, J. PhotochemCAD Spectra by Category. Available at:
http://omic.ogi.edu/spectra/PhotochemCAD/hmtl/.