Supplementary Materials:
Conformational stability of CopC and roles of residues Tyr79 and Trp83
Zhen Song, Xiaoyan Zheng, BinSheng Yang*
Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering
of Ministry of Education, Shanxi University, Taiyuan, CHINA, 030006
Corresponding author (email: yangbs@sxu.edu.cn Tel.: 0351-7016358)
1. The method of calculation of the binding constants
In order to calculate the binding constant between Cu(II) and mutants, we used
EDTA as a competitive ligands. Titration of apoCopC and its mutants with EDTACu(II) give the value of fluorescence intensity at 320 nm, titration curves were
prepared by plotting F vs. [Cu(II)]/[protein]. Assuming that the decrease of
fluorescence quench at given [Cu(II)]/[protein] is attribute to the change of
Cu2+-protein to Cu2+-EDTA. The binding constant can be calculated with the
following Formulas (1) to (7). The binding constants of Cu2+ and proteins were list in
Table 4.
Protein＋Cu(II)  Cu(II)– Protein
[EDTA − Cu(II)] =
Fb −Fa
F0 −F∞
(1)
[Protein]t
(2)
[EDTA]f = [EDTA]t − [EDTA − Cu(II)]
[Cu(II)]f = K
[EDTA−Cu(II)]
(4)
Protein−Cu(II) ·[EDTA]f
[Protein − Cu(II)] =
F0 −Fb
F0 −F∞
[Protein]t
[Protein]f = [Protein]t − [Protein − Cu(II)]
K Protein−Cu(II) =
[Protein−Cu(II)]
[Protein]f [Cu(II)]f
(3)
(5)
(6)
(7)
Where [Cu(Ⅱ)-Protein], [Protein]f, [Cu(Ⅱ)-EDTA] and [EDTA]f represent
the concentration of complex [Cu(Ⅱ)-Protein], free protein, the complex
[Cu(Ⅱ)-EDTA] and free EDTA, respectively. [EDTA]t and [Protein]t are the total
concentration of EDTA and proteins,
respectively. F(a) and F(b) represent the
fluorescence intensity of proteins when EDTA is absent and present, respectively. F∞
is the fluorescence intensity of [Cu(Ⅱ)-Protein]. F0 is the initial fluorescence
intensity of Protein.
2. Analysis of denaturation data
A new model used to calculate the free energy change of protein unfolding is
proposed some time ago.26 In this model, it is considered that proteins are composed
of structural elements. The unfolding of a structural element obeys a two-state
mechanism and the free energy change of the element can be obtained by a linear
extrapolation method.
A structural element, Ei can change from a native state to a denatured, unfolded
state. The dependence of ΔGi on denaturant concentration [D] is shown in eq. (8):
∆G𝑖 = −RT ln k 𝑖 = ∆G𝑖0 (H2 O) + m𝑖 [D]
(8)
If the number of structural elements is n in a protein and structural element 1
appears n1 times, structural element 2 appears n2 times, and structural element k
appears nk times, the unfolding curve can be fitting by eq. (9):
𝑖
Yapp = ∑k𝑖=1 f𝑖 ∙ Yapp
Where f𝑖 = n𝑖 /n and
(9)
∑k𝑖=1 n𝑖 = n, ni is the number of Ei appearing times in
the protein, and
∑k𝑖=1 f𝑖 = 1
(10)
The free energy change of the secondary structure for a protein is the sum of
those for all of the structural elements.
0
∆Gprotein
(H2 O) = ∑𝑘𝑖=1 n𝑖 ∙ ∆G𝑖0 (H2 O)
(11)
The average structural element free energy change can be calculated using eq.
(12):
0
0
(H2 O) >= ∑𝐾
< ∆Gelement
𝑖=1 f𝑖 ∙ ∆G𝑖 (H2 O)
(12)
Figure S1. Fluorescence spectra for Y79F under different GdnHCl concentration. The
concentration of GdnHCl from a to g is 0, 0.5, 1.2, 1.5, 1.8, 2.0, 2.5 M, respectively.
3. Multi-state free energy change
The results from the experiments suggested that there may be two different
structural elements in Y79W and Y79WW83F, one is structural element 1 and another
is structural element 2, average structural element free energy change can be
calculated from eq 13.
0
(H2 O) >= f1 ∙ ∆G10 (H2 O) + f2 ∙ ∆G20 (H2 O)
< ∆Gelement
(13)
The urea-induced unfolding profiles of both Y79W and Y79WW83F show
three-state processes (Figs. S2 and S3), and the GdnHCl-induced unfolding profiles of
Y79WW83F also shows three-state processes (Fig. S4).
Figure S2. Urea induced unfolding of Y79W (●). Data are fitted by using eq. (9).
0
(H2 O) >= 0.87∆G10 (H2 O) + 0.13∆G20 (H2 O)
< ∆Gelement
Figure S3. Urea induced unfolding of Y79WW83F (●). Data are fitted by using eq.
(9).
0
(H2 O) >= 0.75∆G10 (H2 O) + 0.24∆G20 (H2 O)
< ∆Gelement
Figure S4. GdnHCl induced unfolding of Y79WW83F (●). Data are fitted by using eq.
(9).
0
(H2 O) >= 0.69∆G10 (H2 O) + 0.31∆G20 (H2 O)
< ∆Gelement
4. The NMR structure of apoCopC (1M42)
Figure S5 A. Segment of apoCopC encompassing residues 69-81 of strands 5 and 6.
The hydrogen bond between the hydroxyl group of Tyr79 and the backbone carbonyl
group of Thr75 (the tyrosine corner) is shown (PDB: 1M42). B. Comparison of
apoCopC (Tyr79 in green) and Y79F (Phe79 in cyan). C. Comparison of apoCopC
(Tyr79 in green) and Y79W (Trp79 in purple). D. Comparison of Y79W (Trp83 in
blue) and Y79WW83F (Phe83 in black), Y79WW83L (Leu83 in yellow). The high
hydrophobic region was color in red. The structures in the dished line clearly show
the ellipse area in the figure. The structures of mutants come from Phyre. All protein
structure displays were created with PyMOL.