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

Summary

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.

Abstract

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.

Introduction

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 m....

Protocol

1. Sample Preparation

1. Apo-sample: Dissolve approximately 1-2 mg of the peptide in 450-500 µl of deuterated NMR grade solvent (the preferable solutions for biological samples are 10% D2O in water or d6-DMSO^11-12 if the sample does not dissolve in water or reacts with it).
2. Copper-reacted sample: Dissolve approximately 1-2 mg of the peptide with an equimolar amount of metal salt in 450-500 µl of the NMR solvent.
3. Filter the solution using a Sinter glass, filtration paper or any other technique that suits the compounds under investigation and does not adsorb them. This is essential to remove any metall....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Avance DMX 600 MHz Spectrometer	Bruker
NMR sample tubes	Wilmad	535-PP
Glove box	MBraun	LM05-019
Lyophilizer	VirTis	benchtopK
Peptide	BioChemia	Custom made	>95% purity
Copper (1) chloride	Aldrich	224332
Hydrochloric acid	BioLab	231-595-7
Sodium hydroxide	Gadot	1310-73-2
d6-Dimethylsulfoxide	Aldrich	236926
Deuterium oxide	Aldrich	151882