Solution Structure of the Circular γ-Domain Analog from the Wheat Metallothionein Ec-1
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design, Purity, and Metal Ion Binding Abilities of the Circular γ-Ec-1 Analog
2.2. pH Stability of the Metal-Thiolate Clusters
Equation 1 | cyc-Zn2γ-Ec-1 | lin-Zn2γ-Ec-1 | cyc-Cd2γ-Ec-1 | lin-Cd2γ-Ec-1 |
---|---|---|---|---|
AMT | 18062 ± 67 | 17696 ± 261 | 19981 ± 109 | 20539 ± 200 |
AMTHn | −295 ± 82 | −161 ± 372 | 116 ± 101 | −395 ± 361 |
pK | 4.287 ± 0.003 | 4.26 ± 0.03 | 3.444 ± 0.003 | 3.59 ± 0.02 |
n | 3.85 ± 0.10 | 1.6 ± 0.1 | 4.5 ± 0.1 | 1.9 ± 0.1 |
Equation 2 | ||||
AMT | 18504 ± 141 | 20979 ± 61 | ||
A MTHm | 11518 ± 1061 | 13634 ± 1174 | ||
A MTHn+m | 176 ± 172 | −109 ± 124 | ||
pK1 | 4.129 ± 0.010 | 3.466 ± 0.007 | ||
pK2 | 4.9 ± 0.2 | 4.2 ± 0.1 | ||
n | 4.3 ± 0.5 | 3.9 ± 0.3 | ||
m | 0.9 ± 0.1 | 1.1 ± 0.1 |
2.3. Structural Analysis with NMR Spectroscopy
2.3.1. Conformational Constraints and Structure Calculation
NOE restraints | ||
---|---|---|
Total | 201 | |
Sequential (|i-j| < 1) | 142 | |
Medium (1 < |i-j| <5) | 38 | |
Long range (|i-j| <5) | 21 | |
RMSD to average structure, Å (res. 2-23) Before water refinement | After water refinement | |
Backbone (N,Cα,C) | 0.71 | 0.80 |
All heavy atoms | 1.03 | 1.10 |
Number of close contacts(within 1.6 Å for H atoms, 2.2 Å for heavy atoms) | 18 | 0 |
RMS deviation for bond angles (°) | 0.2 | 1.4 |
RMS deviation for bond lengths(Å) | 0.001 | 0.016 |
Ordered residues | 3A-14A | 2A-23A |
Procheck-NMR (residues 2-23) [30] | ||
In most favored regions (%) | 58,3 | 73.9 |
In additional allowed (%) | 41.7 | 26.1 |
In generously allowed (%) | 0 | 0 |
In disallowed regions (%) | 0 | 0 |
Verify3D (residues 2-23) [31] | ||
Raw score | 0.43 | 0.5 |
Z-score | −0.48 | 0.64 |
2.3.2. Comparison of the cyc-γ-Ec-1 Structure with its Linear Analog
3. Experimental
3.1. Chemicals and Solutions
3.2. Expression and Purification of cyc-γ-Ec-1
3.3. Preparation of cyc-Zn2γ−Ec-1 and cyc-Cd2γ−Ec-1
3.4. Mass Spectrometry
3.5. UV/Vis, CD, and MCD Spectroscopy
3.6. pH Titrations
3.7. NMR Spectroscopy
3.8. Structure Calculation
4. Conclusions
Supplementary Materials
Acknowledgments
Conflicts of Interest
References
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Tarasava, K.; Johannsen, S.; Freisinger, E. Solution Structure of the Circular γ-Domain Analog from the Wheat Metallothionein Ec-1. Molecules 2013, 18, 14414-14429. https://doi.org/10.3390/molecules181114414
Tarasava K, Johannsen S, Freisinger E. Solution Structure of the Circular γ-Domain Analog from the Wheat Metallothionein Ec-1. Molecules. 2013; 18(11):14414-14429. https://doi.org/10.3390/molecules181114414
Chicago/Turabian StyleTarasava, Katsiaryna, Silke Johannsen, and Eva Freisinger. 2013. "Solution Structure of the Circular γ-Domain Analog from the Wheat Metallothionein Ec-1" Molecules 18, no. 11: 14414-14429. https://doi.org/10.3390/molecules181114414