Experimental Insight into the Structural and Functional Roles of the ‘Black’ and ‘Gray’ Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs
Abstract
:1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Materials
3.2. Expression and Purification of Human Wild-Type Recoverin and Its Mutants
3.3. Removal of Metal Ions from Recoverin
3.4. Chemical Crosslinking of Recoverin and Its Mutants
3.5. Circular Dichroism Measurements
3.6. Fluorescence Measurements
3.7. Evaluation of Calcium Binding Parameters by Means of The Calcium Buffer Method
α = 1/(1 + f1 + f1 × f2),
f1 = 10(pK1 - pCa),
f2 = 10(pK2 - pCa).
f = 10[(logK − pM) × n].
3.8. Determination of Metal Affinity of Recoverin from Protein Titration by Ca2+
P + M ↔ P∙M,
Ka2
P∙M + M ↔ P∙M2,
Protein + M ↔ Protein∙M,
3.9. The Equilibrium Centrifugation Assay of Recoverin Binding to Membranes
3.10. Rhodopsin Phosphorylation Assay
3.11. Computational Evaluation of the Intrinsic Disorder Predisposition of Human Recoverin and Its Cluster Mutants
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples of the compounds wild type recombinant recoverin and its mutants are available from the authors. |
Protein | Protein State | α-Helices % | β-Sheets % | Turns % | Unordered Structure | ||
---|---|---|---|---|---|---|---|
rWT | Apo-form | 48.1 ± 0.1 | 8.9 ± 0.3 | 16.5 ± 0.2 | 26.8 ± 0.3 | ||
N-domain | ‘black’ cluster | F35A | 47.9 ± 0.2 | 8.6 ± 0.4 | 16.6 ± 0.2 | 26.7 ± 0.3 | |
F83A | 49.3 ± 0.3 | 7.5 ± 0.3 | 15.9 ± 0.2 | 27.0 ± 0.1 | |||
Y86A | 48.5 ± 0.2 | 8.0 ± 0.2 | 15.7 ± 0.2 | 27.9 ± 0.3 | |||
‘gray’ cluster | F70A | 47.9 ± 0.3 | 8.4 ± 0.3 | 16.1 ± 0.3 | 27.6 ± 0.3 | ||
Q46A | 48.1 ± 0.4 | 7.9 ± 0.2 | 15.7 ± 0.2 | 27.8 ± 0.1 | |||
F49A | 46.4 ± 0.5 | 8.9 ± 0.3 | 16.4 ± 0.4 | 28.5 ± 0.5 | |||
C-domain | ‘black’ cluster | F106A | 49.7 ± 0.1 | 7.6 ± 0.4 | 15.8 ± 0.1 | 26.8 ± 0.4 | |
E169A | 46.9 ± 0.9 | 8.7 ± 0.3 | 17.4 ± 0.2 | 27.3 ± 0.7 | |||
F172A | 50.0 ± 0.3 | 7.9 ± 0.2 | 16.4 ± 0.4 | 25.3 ± 0.3 | |||
‘gray’ cluster | W156A | 47.0 ± 0.3 | 9.0 ± 0.3 | 17.0 ± 0.1 | 27.4 ± 0.1 | ||
K119A | 49.2 ± 0.3 | 8.1 ± 0.3 | 16.1 ± 0.2 | 26.4 ± 0.2 | |||
V122A | 47.1 ± 0.2 | 8.9 ± 0.3 | 16.4 ± 0.1 | 27.9 ± 0.2 | |||
rWT | Mg2+ | 49.3 ± 0.1 | 7.9 ± 0.3 | 17.9 ± 0.2 | 25.3 ± 0.3 | ||
N-domain | ‘black’ cluster | F35A | 49.9 ± 0.2 | 8.2 ± 0.4 | 16.8 ± 0.2 | 25.4 ± 0.3 | |
F83A | 51.1 ± 0.3 | 7.1 ± 0.3 | 15.9 ± 0.2 | 26.0 ± 0.1 | |||
Y86A | 50.9 ± 0.2 | 7.3 ± 0.2 | 16.6 ± 0.2 | 25.3 ± 0.3 | |||
N-domain | ‘gray’ cluster | F70A | 48.2 ± 0.3 | 8.0 ± 0.3 | 17.4 ± 0.3 | 26.6 ± 0.3 | |
Q46A | 50.9 ± 0.4 | 7.8 ± 0.2 | 16.0 ± 0.2 | 25.3 ± 0.1 | |||
F49A | 48.4 ± 0.5 | 7.9 ± 0.3 | 16.8 ± 0.4 | 27.1 ± 0.5 | |||
C-domain | ‘black’ cluster | F106A | 53.2 ± 0.1 | 7.0 ± 0.4 | 16.2 ± 0.1 | 24.2 ± 0.4 | |
E169A | 49.8 ± 0.9 | 7.4 ± 0.3 | 17.1 ± 0.2 | 26.2 ± 0.7 | |||
F172A | 51.1 ± 0.3 | 7.9 ± 0.2 | 15.7 ± 0.4 | 25.5 ± 0.3 | |||
‘gray’ cluster | W156A | 49.1 ± 0.3 | 7.4 ± 0.3 | 17.4 ± 0.1 | 26.3 ± 0.1 | ||
K119A | 51.3 ± 0.3 | 7.8 ± 0.3 | 15.5 ± 0.2 | 25.4 ± 0.2 | |||
V122A | 48.9 ± 0.2 | 7.9 ± 0.3 | 16.7 ± 0.1 | 26.5 ± 0.2 | |||
rWT | Ca2+ | 51.2 ± 0.1 | 7.0 ± 0.3 | 16.0 ± 0.2 | 25.7 ± 0.3 | ||
N-domain | ‘black’ cluster | F35A | 55.0 ± 0.2 | 7.5 ± 0.4 | 14.3 ± 0.2 | 23.5 ± 0.3 | |
F83A | 57.4 ± 0.3 | 5.4 ± 0.3 | 14.4 ± 0.2 | 22.5 ± 0.1 | |||
Y86A | 56.9 ± 0.2 | 5.4 ± 0.2 | 13.9 ± 0.2 | 23.5 ± 0.3 | |||
‘gray’ cluster | F70A | 52.0 ± 0.3 | 7.0 ± 0.3 | 15.8 ± 0.3 | 25.1 ± 0.3 | ||
Q46A | 54.6 ± 0.4 | 7.0 ± 0.2 | 15.4 ± 0.2 | 23.0 ± 0.1 | |||
F49A | 53.9 ± 0.5 | 6.9 ± 0.3 | 15.9 ± 0,4 | 22.8 ± 0.5 | |||
C-domain | ‘black’ cluster | F106A | 55.7 ± 0.1 | 5.8 ± 0.4 | 14.0 ± 0.1 | 24.2 ± 0.4 | |
E169A | 52.3 ± 0.9 | 6.8 ± 0.3 | 15.3 ± 0.2 | 25.3 ± 0.7 | |||
F172A | 54.2 ± 0.3 | 6.6 ± 0.2 | 15.5 ± 0.4 | 23.4 ± 0.3 | |||
‘gray’ cluster | W156A | 52.4 ± 0.3 | 7.1 ± 0.3 | 15.5 ± 0.1 | 24.9 ± 0.1 | ||
K119A | 54.4 ± 0.3 | 6.8 ± 0.3 | 15.4 ± 0.2 | 23.5 ± 0.2 | |||
V122A | 57.6 ± 0.2 | 6.0 ± 0.3 | 14.4 ± 0.1 | 22.0 ± 0.2 |
Protein | Protein State | Monomer 20 kDa, % | Dimer 50 kDa, % | Trimer 70–100 kDa, % | Multimer 100–250 kDa, % | ||
---|---|---|---|---|---|---|---|
rWT | Apo-state | 66.9 ± 1.1 | 33.1 ± 1.1 | - | - | ||
N-domain | ‘black’ cluster | F35A | 62.8 ± 0.5 | 37.2 ± 0.5 | - | - | |
F83A | 64.4 ± 0.8 | 35.6 ± 0.8 | - | - | |||
Y86A | 69.3 ± 3.0 | 30.7 ± 3.0 | - | - | |||
‘gray’ cluster | F70A | 70.7 ± 1.7 | 29.3 ± 1.7 | - | - | ||
Q46A | 64.9 ± 1.8 | 35.1 ± 1.8 | - | - | |||
F49A | 63.3 ± 1.0 | 36.7 ± 1.0 | - | - | |||
C-domain | ‘black’ cluster | F106A | 62.0 ± 0.3 | 38.0 ± 0.3 | - | - | |
E169A | 61.1 ± 1.2 | 38.9 ± 1.2 | - | - | |||
F172A | 66.4 ± 2.0 | 33.6 ± 2.0 | - | - | |||
‘gray’ cluster | W156A | 64.6 ± 0.8 | 35.4 ± 0.8 | - | - | ||
K119A | 63.3 ± 0.8 | 36.7 ± 0.8 | - | - | |||
V122A | 73.0 ± 1.2 | 27.0 ± 1.2 | - | - | |||
rWT | Mg2+-loaded | 68.45 ± 0.09 | 31.55 ± 0.09 | - | - | ||
N-domain | ‘black’ cluster | F35A | 69.1 ± 1.9 | 30.9 ± 1.9 | - | ||
F83A | 66.2 ± 1.0 | 33.8 ± 1.0 | - | ||||
Y86A | 66.1 ± 0.2 | 33.9 ± 0.2 | - | ||||
‘gray’ cluster | F70A | 73.1 ± 0.9 | 26.9 ± 0.9 | - | - | ||
Q46A | 69.8 ± 0.5 | 30.2 ± 0.5 | - | - | |||
F49A | 67.9 ± 0.5 | 32.1 ± 0.5 | - | - | |||
C-domain | ‘black’ cluster | F106A | 64.6 ± 1.0 | 35.4 ± 1.0 | - | - | |
E169A | 66.3 ± 0.5 | 33.7 ± 0.5 | - | - | |||
F172A | 63.4 ± 0.7 | 36.6 ± 0.7 | - | - | |||
‘gray’ cluster | W156A | 63.2 ± 0.2 | 36.8 ± 0.2 | - | - | ||
K119A | 53.6 ± 5.0 | 46.4 ± 5.0 | - | - | |||
V122A | 64.7 ± 0.9 | 35.3 ± 0.9 | - | - | |||
rWT | Ca2+-loaded | 15.5 ± 0.6 | 30.9 ± 0.4 | 32.0 ± 1.1 | 21.55 ± 0.09 | ||
N- domain | ‘black’ cluster | F35A | 37.4 ± 3.6 | 31.7 ± 0.3 | 17.78 ± 0.07 | 13.2 ± 3.3 | |
F83A | 38.4 ± 1.4 | 28.7 ± 0.8 | 19.7 ± 0.4 | 13.2 ± 0.3 | |||
Y86A | 35.8 ± 2.7 | 28.5 ± 1.4 | 21.5 ± 0.7 | 14.2 ± 0.6 | |||
‘gray’ cluster | F70A | 40.1 ± 1.8 | 29.3 ± 1.1 | 19.1 ± 0.5 | 11.4 ± 0.2 | ||
Q46A | 31.9 ± 1.9 | 27.3 ± 1.5 | 22.3 ± 1.0 | 18.4 ± 0.6 | |||
F49A | 30.6 ± 1.6 | 25.5 ± 0.5 | 22.8 ± 1.3 | 21.08 ± 0.09 | |||
C-domain | ‘black’ cluster | F106A | 17.9 ± 2.1 | 27.11 ± 0.08 | 30.0 ± 1.4 | 25.0 ± 0.8 | |
E169A | 16.1 ± 1.5 | 27.0 ± 0.3 | 29.6 ± 0.3 | 27.2 ± 0.9 | |||
F172A | 22.0 ± 0.9 | 29.3 ± 0.2 | 28.8 ± 0.8 | 20.0 ± 0.3 | |||
‘gray’ cluster | W156A | 20.4 ± 0.7 | 25.0 ± 1.1 | 24.8 ± 1.0 | 29.8 ± 0.9 | ||
K119A | 19.5 ± 0.4 | 24.4 ± 0.6 | 24.7 ± 0.2 | 31.38 ± 0.05 | |||
V122A | 15.8 ± 0.2 | 27.1 ± 1.3 | 30.3 ± 0.6 | 26.7 ± 2.1 |
Protein | T1/2, °C | Protein | T1/2, °C | ||||
---|---|---|---|---|---|---|---|
N-terminal domain | ‘black cluster’ | rWT | 64.3 ± 2.5 | C-terminal domain | ‘black cluster’ | ||
F35A | 59.8 ± 1.2 | F106A | 61.2 ± 0.6 | ||||
F83A | 56.8 ± 0.1 | E169A | 62.6 ± 3.5 | ||||
Y86A | 67.8 ± 1.3 | F172A | 53.4 ± 0.2 | ||||
‘gray cluster’ | F70A | 66.5 ± 1.4 | ‘gray cluster’ | W156A | 48.6 ± 0.8 | ||
Q46A | 60.4 ± 0.5 | K119A | 55.1 ± 0.9 | ||||
F49A | 66.9 ± 1.8 | V122A | 59.0 ± 0.6 |
Protein | Sequential Binding Model | Cooperative Binding Model | |||
---|---|---|---|---|---|
log K1 | log K2 | log K | n | ||
rWT | 4.5 | 4.1 | 4.6 | 1.3 | |
N-terminal domain | |||||
‘black’ cluster | F35A | 4.6 | 4.8 | 4.7 | 1.5 |
F83A | 5.8 | 2.8 | 5,8 | 0.9 | |
Y86A | 6.2 | 4.5 | n/d | n/d | |
‘gray’ cluster | F70A | 3.2 | 3.4 | n/a | n/a |
Q46A | 4.7 | n/a | n/a | ||
F49A | 4.4 | 3.3 | 4.1 | 0.8 | |
C-terminal domain | |||||
‘black’ cluster | F106A | 3.9 | 4.0 | 3.9 | 1.4 |
E169A | 4.4 | 4.1 | 4.6 | 1.4 | |
F172A | n/d | n/d | 4.0 | 1.7 | |
‘gray’ cluster | W156A | n/d | n/d | 3.5 | 1.9 |
K119A | 8.7 | 7.9 | 7.9 | 1.1 | |
V122A | 4.0 | 4.2 | 4.2 | 1.4 |
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Permyakov, S.E.; Vologzhannikova, A.S.; Nemashkalova, E.L.; Kazakov, A.S.; Denesyuk, A.I.; Denessiouk, K.; Baksheeva, V.E.; Zamyatnin, A.A., Jr.; Zernii, E.Y.; Uversky, V.N.; et al. Experimental Insight into the Structural and Functional Roles of the ‘Black’ and ‘Gray’ Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs. Molecules 2019, 24, 2494. https://doi.org/10.3390/molecules24132494
Permyakov SE, Vologzhannikova AS, Nemashkalova EL, Kazakov AS, Denesyuk AI, Denessiouk K, Baksheeva VE, Zamyatnin AA Jr., Zernii EY, Uversky VN, et al. Experimental Insight into the Structural and Functional Roles of the ‘Black’ and ‘Gray’ Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs. Molecules. 2019; 24(13):2494. https://doi.org/10.3390/molecules24132494
Chicago/Turabian StylePermyakov, Sergey E., Alisa S. Vologzhannikova, Ekaterina L. Nemashkalova, Alexei S. Kazakov, Alexander I. Denesyuk, Konstantin Denessiouk, Viktoriia E. Baksheeva, Andrey A. Zamyatnin, Jr., Evgeni Yu. Zernii, Vladimir N. Uversky, and et al. 2019. "Experimental Insight into the Structural and Functional Roles of the ‘Black’ and ‘Gray’ Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs" Molecules 24, no. 13: 2494. https://doi.org/10.3390/molecules24132494