Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and characterize ``invisible'' excited protein states in slow exchange with a visible ground-state conformation (excited-state lifetimes ranging from similar to 5 to 50 ms) is presented. This method, which is an adaptation of the chemical exchange saturation transfer (CEST) magnetic resonance imaging experiment, involves irradiating various regions of the spectrum with a weak B-1 field while monitoring the effect on the visible major-state peaks. The variation in major-state peak intensities as a function of frequency offset and B-1 field strength is quantified to obtain the minor-state population, its lifetime, and excited-state chemical shifts and line widths. The methodology was validated with N-15 CEST experiments recorded on an SH3 domain ligand exchanging system and subsequently used to study the folding transition of the A39G FF domain, where the invisible unfolded state has a lifetime of similar to 20 ms. Far more accurate exchange parameters and chemical shifts were obtained than via analysis of Carr-Purcell-Meiboom-Gill relaxation dispersion data.

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Studying "invisible" excited protein states in slow exchange with a major state conformation.

Paramagnetic metal ions offer outstanding opportunities for protein studies by nuclear magnetic resonance (NMR) spectroscopy. The paramagnetic effects manifested in the NMR spectra provide powerful restraints for the determination of the three-dimensional structure of proteins, open new possibilities for the analysis of protein-protein and protein-ligand interactions, and offer widely applicable tools for sensitivity enhancement of NMR experiments, resonance assignments, and studies of conformational heterogeneity and exchange. With the advent of new reagents for site-specific labeling of proteins with paramagnetic metal ions, a range of established and new strategies that used to be reserved for metalloproteins is becoming available for NMR spectroscopy of nonmetalloproteins.

Protein NMR Using Paramagnetic Ions

Paramagnetic NMR in solution and the solid state

http://www.meilerlab.org/index.php/publications/showPublication/pub_id/100

Expanding the utility of NMR restraints with paramagnetic compounds: background and practical aspects.

Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and long-range structural restraints. PCS are a manifestation of the χ-tensor anisotropy, the Δχ-tensor, which in turn can be determined from the PCS. Once the Δχ-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein–ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Δχ-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Δχ-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits e186 and θ using PCS.

Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts.

The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin–spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin–spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program x-plor and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu–Cys Sγ bond, and a long Cu–Met Sδ bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu(2+) metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and (1)H-(15)N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu(+) plastocyanin and Cd(2+)-substituted plastocyanin as the diamagnetic references, it is found that the Cd(2+)-substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu(2+) plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the (15)N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained (1)H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure.

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On the use of pseudocontact shifts in the structure determination of metalloproteins.

Information about the structure of a native nonmetalloprotein was obtained from the pseudocontact shifts induced by a paramagnetic metal ion bound to the protein. The approach exploits the presence of metal binding sites on the surface of the protein. Using Escherichia coli thioredoxin as a model protein, we show that potential binding sites can be identified using the Cu2+ ion, and that pseudocontact shifts induced by a Ni2+ ion bound to one of these sites can provide valuable long-range structure information about the protein.

Metal−Protein Interactions: Structure Information from Ni2+-Induced Pseudocontact Shifts in a Native Nonmetalloprotein†

A previous method for mapping the electron spin distribution in blue copper proteins by paramagnetic nuclear magnetic resonance (NMR) relaxation (Hansen DF, Led JJ, 2004, J Am Chem Soc 126:1247–1253) suggested that the blue copper site of plastocyanin from Anabaena variabilis (A.v.) is less covalent than those found for other plastocyanins by other experimental methods, such as X-ray absorption spectroscopy. Here, a detailed spectroscopic study revealed that the electronic structure of A.v. plastocyanin is similar to those of other plastocyanins. Therefore, the NMR approach was reinvestigated using a more accurate geometric structure as the basis for the mapping, in contrast to the previous approach, as well as a more complete spin distribution model including Gaussian-type natural atomic orbitals instead of Slater-type hydrogen-like atomic orbitals. The refinement results in a good agreement between the electron spin density derived from paramagnetic NMR and the electronic structure description obtained by the other experimental methods. The refined approach was evaluated against density functional theory (DFT) calculations on a model complex of the metal site of plastocyanin in the crystal phase. In general, the agreement between the experimental paramagnetic relaxation rates and the corresponding rates obtained by the DFT calculations is good. Small deviations are attributed to minor differences between the solution structure and the crystal structure outside the first coordination sphere. Overall, the refined approach provides a complementary experimental method for determining the electronic structure of paramagnetic metalloproteins, provided that an accurate geometric structure is available.

Jens J. Led

Papers

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

On the use of pseudocontact shifts in the structure determination of metalloproteins.

Metal−Protein Interactions: Structure Information from Ni2+-Induced Pseudocontact Shifts in a Native Nonmetalloprotein†

Reinvestigation of the method used to map the electronic structure of blue copper proteins by NMR relaxation.

The Use of NMR in the Studies of Highly Flexible States of Proteins: Relation to Protein Function and Stability