We thank the Human Frontier Science Program (Young investigator grant (RGY)0068-2006) for financial support.

1. Sample Preparation
<ol>
	<li>Apo-sample: Dissolve approximately 1-2 mg of the peptide in 450-500 &#181;l&#160;of deuterated NMR grade solvent (the preferable solutions for biological samples are 10% D2O in water or d6-DMSO11-12 if the sample does not dissolve in water or reacts with it).</li>
	<li>Copper-reacted sample: Dissolve approximately 1-2 mg of the peptide with an equimolar amount of metal salt in 450-500 &#181;l&#160;of the NMR solvent.</li>
	<li>Filter the solution using a Sinter glass,...

<ol>
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The authors declare they have no competing financial interests.

The contribution of structural information to understand binding mechanisms is well-accepted. Peptides are useful as models for protein binding and interactions; however they are not amenable to the main method for structure determination, X-ray crystallography. NMR is particularly useful for these systems, since the structures can be readily solved in solution. This is especially for the case of metallochaperone-mimetics that additionally require structure determination under an inert environment to prevent oxidation of...

Peptides are widely used as protein models, potential drug leads and therapeutic agents in their own right. However, their small size and high degree of flexibility often precludes structure determination by X-ray due to difficulties in crystallization.
Nuclear magnetic resonance (NMR) can be used to determine peptide structures and interactions. The method can give information regarding local and overall structure, binding and lower affinity interactions, and is applicable to difficult samples since it can be done in the solution state.
Copper transport in biological systems is achieved by intracellular copper metallochaperone proteins that specifically bind Cu (I) ions and deliver them to their target proteins through a series of protein-protein interactions, to protect the ions from oxidation and prevent the release of toxic copper2-5. The binding site is characterized by the conserved sequence, MXH/TCXanyXanyC, which has been shown both by NMR and crystallography to bind the Cu (I) by the soft thiolato ligands of the two cysteine residues, although an additional external ligand has also been proposed6-8. The structure-function relationship of these proteins has been a subject of intensive research9.
In the study presented here, a peptide model that includes the conserved sequence of copper metallochaperones was synthesized and reacted with Cu (I) under an inert environment. The presented protocol describes the steps of structure determination by NMR, including sample preparation, data collection, data processing, structure generation and structural analysis. The analysis was done in two steps: First structures were generated with no information regarding the mode of binding of the peptide to the copper ion. Once the binding mode was established empirically, these constraints were introduced to provide a high resolution structure. The mode of binding is the essential point in the model and was thus determined in an unbiased manner.
The NMR structural determination of model peptides is an extremely valuable technique that is often used by chemists and biologists. It may be applied relatively easily to different peptides under different conditions, and thus may shed light on relevant mechanisms10. Understanding the structure elucidation process provide a better understanding of the strengths and weaknesses of the proposed structures.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Avance DMX 600 MHz Spectrometer</td>
			<td style="width:108px;">Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:167px;">NMR sample tubes&#160;</td>
			<td style="width:108px;">Wilmad</td>
			<td style="width:141px;">535-PP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:167px;">Glove box</td>
			<td style="width:108px;">MBraun</td>
			<td style="width:141px;">LM05-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Lyophilizer&#160;&#160;</td>
			<td style="width:108px;">VirTis</td>
			<td style="width:141px;">benchtopK</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Peptide</td>
			<td style="width:91px;">BioChemia</td>
			<td style="width:88px;">Custom made&#160;</td>
			<td style="width:87px;">&#62;95% purity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:136px;">Copper (1) chloride</td>
			<td style="width:91px;">Aldrich</td>
			<td style="width:88px;">224332</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:136px;">Hydrochloric acid</td>
			<td style="width:91px;">BioLab</td>
			<td style="width:88px;">231-595-7</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:136px;">Sodium hydroxide</td>
			<td style="width:91px;">Gadot</td>
			<td style="width:88px;">1310-73-2</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:136px;">d6-Dimethylsulfoxide</td>
			<td style="width:91px;">Aldrich</td>
			<td style="width:88px;">236926</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:136px;">Deuterium oxide</td>
			<td style="width:91px;">Aldrich</td>
			<td style="width:88px;">151882</td>
			<td style="width:87px;"></td>
		</tr>
	</tbody>
</table>

structure and coordination determination of peptide-metal complexes using 1d and 2d 1h nmr

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

As part of an ongoing study of copper-binding protein models, the structure of the conserved sequence MT/HCXXC within the linear peptide MTCSGCSRPG was determined by solution state NMR. The peptide was reacted with CuCl under inert environment and the pH was measured as ~3.0 by a universal stick. The amide region of the peptide showed an expansion upon reaction with copper, from 6.7-8.5 to 6.6-9.0 ppm (Figure 2). Line broadening due to slight copper oxidation is evident in the baseline.
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Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

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9.5K ビュー.  The Hebrew University of Jerusalem. The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.