How many mg2 ions

These results indicate that, in the presence of 4. The changes in the other Ile residues were identical to those in the wild-type protein. Solution NMR method is known as one of the methods to solve the three-dimensional structure of relatively small proteins. For larger proteins, NMR spectral changes caused by the functionally related stimuli such as ligand binding reflect the changes in the conformation and dynamics that are related to their functions.

It should be noted that the structural changes in the TM region seem to occur in two steps Figure 4. The closed gate is formed and stabilized by the interaction between N TM and L Plug on the cytoplasmic side of the ion-conducting pore in the TM region Figure 1—figure supplement 1 Hattori et al.

Thus, the I Plug NMR signal, which is located in the C-terminal region of the plug helix, might serve as a good probe for the conformational change of the gate region. Therefore, the complete conformational change of I Plug is likely to be achieved at 4 mM, which contributes to the stabilization of the closed gate. NMR analysis in combination with a high resolution crystal structure has provided site-specific information on changes in the protein conformation and dynamics of MgtE in relation to its function.

We utilised a plasmid encoding full-length MgtE from T. The MgtE mutant constructs were generated through polymerase chain reaction-based mutagenesis. All MgtE proteins were expressed in E. MgtE and its mutants were purified as follows. The missing signals in the spectra of the Ile mutants were assigned as the signals arising from the mutated Ile residues. The normalised factor 5.

After gigal seal formation, a patch of membrane was excised and the bath solution was exchanged with a batch solution containing mM N -methyl-D-glucamine, 0. Currents were filtered at 2 kHz and sampled at 5 kHz. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Your article has been favorably evaluated by Richard Aldrich Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.

The following individual involved in review of your submission has agreed to reveal their identity: Charles R Sanders Reviewer 2. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision. This is an interesting paper on the role of the binding of seven Mg II ions to the microbial MgtE channel, providing the apparent Mg II binding affinity as well as Hill coefficients informing on binding cooperativity.

This was a technically challenging project in which a combination of sophisticated isotopic labeling methods and methyl-TROSY methods allowed NMR spectra to be acquired for some 15 assigned isoleucine residues spread rather evenly throughout the protein. Different peaks exhibited changes that led to different apparent Kd and Hill coefficients. Additional NMR studies conducted using Mg II ion binding site mutants, led to the realization that three of the Mg II sites form a distinct set of sites from the other 4 sites.

In particular, the three sites are proposed to be responsible for stabilizing the open form of the channel, whereas the other 4 may help stabilize the channel but also exert other modulating effects, perhaps leading to channel closure at higher ion concentrations. The reviewers agreed that the work is interesting and important. However, they felt that relating Mg II binding results to function would be critical to establish the role of the bound ions.

In particular, it was pointed out that the binding data and structural inferences that suggest highly cooperative ion binding should be reconciled with existing functional data e. Furthermore, similar functional data on mutants targeting Mg II binding sites should help resolve the role of the groups of the sites. For example, mutations of sites 4, 5, and 7 should lead to a channel that does not close at elevated Mg II concentrations.

The manuscript by Osawa et al. The channel is tightly regulated by Mg, and closes at Mg concentrations above 5 mM. Previous crystal structures showed that the dimeric channel contains one central Mg ion in the permeation pathway Mg1 and 6 additional sites per subunit in three cytoplasmic regions: N-domain, CBS domain and plug helix.

They found that in the absence of Mg, N-domain and plug residue I showed high mobility. Titrating in Mg, led to changes of Ile peaks characteristics throughout the protein with EC50 varying between 0. The authors mutated out each Mg binding site one at a time, and found Mg binding to sites Mg1, 2, 3 and 6 was essential for all conformational changes.

In contrast, binding to sites Mg4, 5, and 7 was not essential for changes in TM regions, but was required for changes in N-domain, CBS domain, and plug helix Mg 5 and 7. The authors construct a model whereby at low, but physiologically relevant Mg concentrations, Mg binds cooperatively to sites 1, 2, 3 and 6 to produce a conducting state; at higher concentrations Mg binds cooperatively to sites 4, 5 and 7, increasing interactions between N-domain and CBS and leading to channel closing.

I found the work compelling even if there were some unresolved questions. The authors should discuss what are the differences and what might be their origin.

The last term is ambiguous here. The latter residue shows sensitivity more similar to residues in the N- and CBS-domains. Therefore, while Mg binding to the sites might be cooperative, the structural changes in TM region may occur in two steps with binding of Mg to the first and second sets of binding sites. This needs to be clarified. Binding to site 4 does not seem to affect the structural changes in the plug, while binding to sites 5 and 7 does.

Along with the results of additional NMR studies conducted using Mg II ion binding site mutant, this led to the realization that three of the Mg II sites can regarded a distinct set of site from the other 4 sites. Exactly what these other roles is was a little vague. This is an admirable and difficult study that significantly advances our understanding of Mg II channel structure and function.

This well written paper could be published essentially as is. However, the degree to which this NMR study established exactly what these two sets of Mg II binding events are doing in terms of channel function would be greatly enhanced by accompanying [Mg II ]-dependent channel function measurements for both WT and the mutants they characterized by NMR. More Chemistry Questions Q1. Which of the following pair is correct?

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Which among the following is called the 'Fool's Gold'? Chen Z. Phillips J. Scalable molecular dynamics with NAMD. MacKerell A. Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations.

Saladin A. PTools: an open source molecular docking library. BMC Struct. Haruta N. Ito K. Two three-strand intermediates are processed during Raddriven DNA strand exchange. Bazemore L. Kinetic analysis of pairing and strand exchange catalyzed by RecA. Detection by fluorescence energy transfer. Story R. The structure of the E. Sancar A. Sequences of the recA gene and protein. Daniel J.

Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry. Mass Spectrom. Aihara H. An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing. Yang D. Feng B. Evidence for hydrophobic catalysis of DNA strand exchange. Bugreev D. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.

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