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Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k
Lena Mäler , John Blankenship, Mark Rance and Walter J. Chazin*
Supplementary Material
Table S1. Precision of the N56A structure and comparison to the apo and (Ca2+)2 state.a
24 N56A structures N56A vs apob N56A vs (Ca2+)2b
|
Residuesc |
All heavy atoms |
Backbone |
Backbone |
Backbone |
|
All |
1.20 ± 0.11 |
0.66 ± 0.11 |
2.32 ± 0.17 |
2.34 ± 0.23 |
|
All helices |
0.92 ± 0.08 |
0.30 ± 0.05 |
1.16 ± 0.13 |
0.80 ± 0.11 |
|
Helix I/II |
0.85 ± 0.09 |
0.19 ± 0.05 |
0.86 ± 0.14 |
0.59 ± 0.07 |
|
Helix III/IV |
0.95 ± 0.10 |
0.29 ± 0.07 |
1.18 ± 0.17 |
0.74 ± 0.10 |
a
All values reported as average rmsd from the mean structure.b
The ensemble of N56A structures compared to the ensembles of 33 apo and 31 (Ca2+)2 calbindin D9k structures.c
The residues used for calculating rmsds are : all; 1-75; helix I, 3-15; helix II, 25-35; helix III, 46-53; helix IV, 63-73.
Table S2. Model-free parameters for the (Ca2+)1 state of N56A calbindin D9k.
|
Residue |
Modela |
S2 |
t e (ps) |
Rex(s-1) |
|
Ser2 |
2 |
0.74 ± 0.01 |
96 ± 14 |
|
|
Glu4 |
2 |
0.82 ± 0.01 |
63 ± 19 |
|
|
Glu5 |
2 |
0.86 ± 0.01 |
140 ± 35 |
|
|
Leu6 |
1 |
0.85 ± 0.01 |
||
|
Lys7 |
2 |
0.87 ± 0.01 |
39 ± 26 |
|
|
Gly8 |
1 |
0.87 ± 0.01 |
||
|
Ile9 |
2 |
0.85 ± 0.01 |
51 ± 22 |
|
|
Phe10 |
2 |
0.87 ± 0.01 |
34 ± 20 |
|
|
Glu11 |
1 |
0.89 ± 0.01 |
||
|
Lys12 |
1 |
0.85 ± 0.01 |
||
|
Tyr13 |
2 |
0.84 ± 0.01 |
35 ± 21 |
|
|
Ala14 |
2 |
0.86 ± 0.01 |
34 ± 25 |
|
|
Ala15 |
1 |
0.86 ± 0.01 |
||
|
Lys16 |
2 |
0.82 ± 0.01 |
16 ± 9 |
|
|
Glu17 |
2 |
0.85 ± 0.01 |
23 ± 20 |
|
|
Gly18 |
1 |
0.90 ± 0.01 |
||
|
Asp19 |
nd |
|||
|
Asn21 |
1 |
0.86 ± 0.01 |
||
|
Gln22 |
1 |
0.86 ± 0.01 |
||
|
Leu23 |
1 |
0.88 ± 0.01 |
||
|
Ser24 |
1 |
0.89 ± 0.01 |
||
|
Lys25 |
1 |
0.87 ± 0.01 |
||
|
Glu26 |
2 |
0.86 ± 0.01 |
29 ± 20 |
|
|
Glu27 |
2 |
0.86 ± 0.01 |
36 ± 19 |
|
|
Leu28 |
1 |
0.88 ± 0.01 |
||
|
Lys29 |
1 |
0.87 ± 0.01 |
||
|
Leu30 |
1 |
0.87 ± 0.01 |
||
|
Leu31 |
1 |
0.90 ± 0.01 |
||
|
Leu32 |
1 |
0.89 ± 0.01 |
||
|
Gln33 |
1 |
0.88 ± 0.01 |
||
|
Thr34 |
1 |
0.88 ± 0.01 |
||
|
Glu35 |
1 |
0.88 ± 0.01 |
||
|
Phe36 |
1 |
0.83 ± 0.01 |
||
|
Ser38 |
2 |
0.85 ± 0.01 |
35 ± 19 |
|
|
Leu39 |
2 |
0.83 ± 0.01 |
64 ± 19 |
0.46 ± 0.35 |
|
Leu40 |
2 |
0.81 ± 0.01 |
60 ± 16 |
|
|
Lys41 |
2 |
0.68 ± 0.02 |
75 ± 15 |
|
|
Gly42 |
2 |
0.56 ± 0.01 |
86 ± 9 |
|
|
Met43 |
nd |
|||
|
Ser44 |
4 |
0.64 ± 0.01 |
70 ± 9 |
0.54 ± 0.29 |
|
Thr45 |
2 |
0.77 ± 0.01 |
87 ± 14 |
|
|
Leu46 |
2 |
0.78 ± 0.01 |
93 ± 15 |
|
|
Asp47 |
1 |
0.84 ± 0.01 |
||
|
Glu48 |
1 |
0.86 ± 0.01 |
||
|
Leu49 |
2 |
0.84 ± 0.01 |
56 ± 19 |
|
|
Phe50 |
2 |
0.88 ± 0.01 |
59 ± 24 |
|
|
Glu51 |
2 |
0.84 ± 0.01 |
26 ± 16 |
|
|
Glu52 |
2 |
0.84 ± 0.01 |
36 ± 18 |
|
|
Leu53 |
1 |
0.84 ± 0.01 |
||
|
Asp54 |
2 |
0.83 ± 0.01 |
40 ± 21 |
|
|
Lys55 |
4 |
0.86 ± 0.01 |
31 ± 20 |
0.37 ± 0.32 |
|
Ala56 |
1 |
0.87 ± 0.01 |
||
|
Gly57 |
2 |
0.77 ± 0.01 |
53 ± 13 |
|
|
Asp58 |
2 |
0.80 ± 0.01 |
62 ± 16 |
|
|
Gly59 |
nd |
|||
|
Glu60 |
2 |
0.81 ± 0.01 |
54 ± 20 |
|
|
Val61 |
2 |
0.79 ± 0.01 |
24 ± 14 |
|
|
Ser62 |
1 |
0.84 ± 0.01 |
||
|
Phe63 |
1 |
0.89 ± 0.01 |
||
|
Glu64 |
2 |
0.83 ± 0.01 |
45 ± 22 |
|
|
Glu65 |
1 |
0.86 ± 0.01 |
||
|
Phe66 |
1 |
0.87 ± 0.01 |
||
|
Gln67 |
1 |
0.87 ± 0.01 |
||
|
Val68 |
1 |
0.85 ± 0.01 |
||
|
Leu69 |
1 |
0.82 ± 0.01 |
||
|
Val70 |
1 |
0.83 ± 0.01 |
||
|
Lys71 |
2 |
0.86 ± 0.01 |
35 ± 20 |
|
|
Lys72 |
nd |
|||
|
Ile73 |
2 |
0.79 ± 0.01 |
64 ± 14 |
|
|
Ser74 |
2 |
0.77 ± 0.01 |
76 ± 13 |
|
|
Gln75b |
5 |
0.09 ± 0.01 |
1547 ± 219 |
a
Dynamical model used to fit the relaxation data. For details see Methods section. The notation "nd" indicates that the relaxation parameters could not be determined.b
For residue 75 S2=S2f*S2s. and te=ts as described in the Methods section.
Table S3. Average order parameters for different structural elements in calbindin D9ka.
Average S2
|
Structural elementb |
apo |
N56A (Ca2+)1 |
(Cd2+)1 |
(Ca2+)2 |
|
Helix I |
0.85 ± 0.04 |
0.85 ± 0.02 |
0.85 ± 0.03 |
0.85 ± 0.04 |
|
Loop I |
0.79 ± 0.04 |
0.86 ± 0.03 |
0.81 ± 0.02 |
0.83 ± 0.03 |
|
Helix II |
0.87 ± 0.03 |
0.86 ± 0.01 |
0.88 ± 0.01 |
0.87 ± 0.01 |
|
Helix III |
0.82 ± 0.01 |
0.84 ± 0.03 |
0.86 ± 0.03 |
0.84 ± 0.01 |
|
Loop II |
0.72 ± 0.11 |
0.82 ± 0.03 |
0.86 ± 0.03 |
0.83 ± 0.03 |
|
Helix IV |
0.86 ± 0.06 |
0.85 ± 0.03 |
0.85 ± 0.05 |
0.82 ± 0.06 |
a
Mean and standard deviation for the generalized order parameter are reported.b
The structural elements are defined: helix I, 4-14; loop I, 16-22; helix II, 25-35; helix III, 46-53; loop II, 55-62; helix IV, 63-73.
Table S4. Ca2+-dependent changes in 15N order parameters and corresponding contributions to changes in backbone conformational entropy in calbindin D9k.
Order Parameters Backbone Entropy Changes
|
Residuea |
apo |
N56A (Ca2+) 1 |
(Cd2+) 1 |
(Ca2+) 2 |
D S Ib (cal-K-1Mol-1) |
D SI Ib (cal-K-1Mol-1) |
||
|
A14 |
0.87 |
0.86 |
0.88 |
0.88 |
||||
|
A15 |
0.83 |
0.86 |
n.d |
0.88 |
* |
|||
|
K16 |
0.82 |
0.82 |
0.78 |
0.80 |
|
+0.45 |
||
|
E17 |
0.78 |
0.85 |
n.dc |
0.83 |
-0.82 |
* |
||
|
G18 |
0.78 |
0.90 |
0.81 |
0.85 |
-1.68 |
(+0.86) |
(-0.50) |
|
|
D19 |
0.73 |
n.d |
0.81 |
0.79 |
* |
-0.77 |
||
|
N21 |
0.82 |
0.86 |
0.83 |
0.86 |
-0.53 |
|||
|
Q22 |
0.83 |
0.86 |
0.84 |
0.83 |
||||
|
L23 |
0.85 |
0.88 |
n.d |
0.84 |
(+0.60) |
* |
||
|
S24 |
0.85 |
0.89 |
n.d |
0.85 |
-0.65 |
(+0.65) |
* |
|
|
K25 |
0.86 |
0.87 |
0.87 |
0.86 |
||||
|
E26 |
0.84 |
0.86 |
0.85 |
0.85 |
||||
|
E27 |
0.88 |
0.86 |
0.88 |
0.87 |
______ |
_____ |
||
|
-3.78 |
(+2.11) |
-0.32 |
(-0.50) |
|||||
|
D54 |
0.80 |
0.83 |
0.88 |
0.85 |
-1.09 |
|||
|
K55 |
0.74 |
0.86 |
0.81 |
0.79 |
-1.33 |
(+0.85) |
-0.69 |
|
|
A56 |
0.80 |
0.87 |
0.85 |
0.85 |
-0.93 |
-0.62 |
||
|
G57 |
0.59 |
0.77 |
0.84 |
0.81 |
-1.32 |
(-0.42) |
-2.12 |
|
|
D58 |
0.63 |
0.80 |
0.86 |
0.87 |
-1.37 |
(-0.93) |
-2.12 |
|
|
G59 |
0.62 |
n.d |
0.90 |
0.86 |
* |
-2.92 |
(+0.72) |
|
|
E60 |
0.69 |
0.81 |
0.88 |
0.85 |
-1.00 |
(-0.97) |
-1.57 |
|
|
V61 |
0.81 |
0.79 |
0.87 |
0.80 |
|
-0.81 |
(+0.93) |
|
|
S62 |
0.90 |
0.84 |
0.90 |
0.85 |
+0.98 |
(+0.86) |
||
|
L63 |
0.88 |
0.89 |
0.88 |
0.86 |
||||
|
E64 |
0.85 |
0.83 |
0.86 |
0.85 |
||||
|
E65 |
0.87 |
0.86 |
0.85 |
0.87 |
______ |
______ |
||
|
-4.97 |
(-1.47) |
-11.94 |
(+2.51) |
|||||
a
The errors for the S2 values are estimated to be ± 0.015 for all states. Only residues in the binding loops for which S2 are significantly different in the Ca2+-bound and apo states are included.b
D SI = SN56A-Sapo and D SII = SCd2+-Sapo. The values in parentheses are for the second binding step along the corresponding binding pathway.c
Order parameters could not be determined due to spetral overlap or substantial broadening of the corresponding resonances.
Last updated January 6, 2000 by Jarrod Smith