Structural investigations of paramagnetic metalloproteins using high-resolution NMR spectroscopy: iron-sulfur and copper proteins

Current approaches to the structural investigations of paramagnetic metal-containing proteins in solution using NMR spectroscopy are surveyed taking iron-sulfur and copper-containing proteins as examples.     

NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors

Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.     

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H₃ (His₃), H₄ (His₄), H₂DH (His₃Asp with two His and one Asp in the same plane) and H₃D (His₃Asp with three His in the same plane) by using the scaffold of the de novo protein GRα₃D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good Cu^II-bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.     

Design of a new mimochrome with unique topology

Peptide-based metalloprotein models represent useful systems to help understand how metalloproteins can support different functions, by the use of similar metal ion cofactors. In order to shed light on the role of the protein matrix in modulating the heme properties, we developed new models: mimochromes. They are pseudo-C(2) symmetric systems, composed of two helical peptides covalently linked to the deuteroporphyrin. The use of C(2) symmetry is particularly advantageous, because it simplifies the design, synthesis and characterization. However, it leaves the problem of possible diastereomeric forms. In the cobalt complex of the first derivative, mimochrome I, Lambda and Delta isomers were indeed experimentally observed. All the insights derived from the Co(III)-mimochrome I structure were used to obtain a re-designed molecule, mimochrome IV. The spectroscopic characterization of the iron and cobalt derivatives suggested the presence of the Lambda isomer as unique species. The NMR solution structure of the diamagnetic Co(III)-mimochrome IV confirmed the ability of the molecule to adopt a unique topology, and revealed the peptide chains to be in helical conformation, as designed. The insertion of intramolecular, inter-chain interactions was successful in favoring the formation of one of the two possible diastereomers. The stereochemically stable structure of mimochrome IV provides an attractive model for modulating the redox potential of the heme, by simple changing the peptide chain composition around the heme.     

Active site comparison of CoII blue and green nitrite reductases

Copper-containing nitrite reductases (NiRs) possess type 1 (T1) and type 2 (T2) copper sites and can be either green or blue in color owing to differences at their T1 centers. The active sites of a green and a blue NiR were studied by utilizing their T1CuI/T2CoII and T1CoII/T2CoII-substituted forms. The UV/Vis spectra of these derivatives highlight the similarity of the T2 centers in these enzymes and that T1 site differences are also present in the CoII forms. The paramagnetic NMR spectra of T1CuI/T2CoII enzymes allow hyperfine shifted resonances from the three T2 His ligands to be assigned: these exhibit remarkably similar positions in the spectra of both NiRs, emphasizing the homology of the T2 centers. The addition of nitrite results in subtle alterations in the paramagnetic NMR spectra of the T1CuI/T2CoII forms at pH<7, which indicate a geometry change upon the binding of substrate. Shifted resonances from all of the T1 site ligands have been assigned and the CoII--N(His) interactions are alike, whereas the CbetaH proton resonances of the Cys ligand exhibit subtle chemical shift differences in the blue and green NiRs. The strength of the axial CoII--S(Met) interaction is similar in the two NiRs studied, but the altered conformation of the side chain of this ligand results in a dramatically different chemical shift pattern for the CgammaH protons. This indicates an alteration in the bonding of the axial ligand in these derivatives, which could be influential in the CuII proteins.     

High-throughput screening of argan oil composition and authenticity using benchtop 1H NMR

We use 60-MHz benchtop nuclear magnetic resonance (NMR) to acquire ¹H spectra from argan oils of assured origin. We show that the low-field NMR spectrum of neat oil contains sufficient information to make estimates of compositional parameters and to inform on the presence of minor compounds. A screening method for quality and authenticity is presented based on nearest-neighbour outlier detection. A variety of oil types are used to challenge the method. In a survey of retail-purchased oils, several instances of fraud were found.     

Deuterium Rotating Frame NMR Relaxation Measurements in the Solid State under Static Conditions for Quantification of Dynamics

The feasibility of static deuterium rotating frame NMR relaxation measurements for characterization of slow timescale motions in powder systems is demonstrated. Using a model compound dimethyl sulfone-d₆, we show that these measurements yield conformational exchange rates and activation energy values in accordance with results obtained with other techniques. Furthermore, we demonstrate that the full Liouvillian approach as opposed to the Redfield approximation is necessary to analyze the experimental data.