Roberta Pierattelli

NMR Spectroscopy of Paramagnetic Metalloproteins

This article deals with the solution structure determination of paramagnetic metalloproteins by NMR spectroscopy. These proteins were believed not to be suitable for NMR investigations for structure determination until a decade ago, but eventually novel experiments and software protocols were developed, with the aim of making the approach suitable for the goal and as user‐friendly and safe as possible. In the article, we also give hints for the optimization of experiments with respect to each particular metal ion, with the aim of also providing a handy tool for nonspecialists. Finally, a section is dedicated to the significant progress made on 13C direct detection, which reduces the negative effects of paramagnetism and may constitute a new chapter in the whole field of NMR spectroscopy.

High‐resolution NMR studies of the zinc‐binding site of the Alzheimer's amyloid β‐peptide

Metal binding to the amyloid β‐peptide is suggested to be involved in the pathogenesis of Alzheimers disease. We used high‐resolution NMR to study zinc binding to amyloid β‐peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo‐form and the holo‐form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N‐relaxation and amide proton exchange experiments on amyloid β‐peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N‐terminus, similar to a previously proposed copper‐binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β‐peptide. The dissociation constant Kd of zinc for the fragment amyloid β‐peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β‐peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal‐induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal‐induced structure of the peptide counteracts aggregation.

Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High-Resolution Proton-Detected Solid-State MAS NMR Spectroscopy

Re-protonation is key: A combination of a high magnetic field (1 GHz) and ultra-fast magic-angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary structures of medium-sized proteins. The technique was applied to the 153-residue microcrystalline Zn II-loaded superoxide dismutase (ZnII-SOD) fully [ 2H,13C,15N]-labeled and 100% re-protonated at the exchangeable sites. Copyright

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15N and 13C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu+ (diamagnetic) or Cu2+ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1H-1H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Carbonic anhydrase inhibitors. Part 37. Novel classes of isozyme I and II inhibitors and their mechanism of action. Kinetic and spectroscopic investigations on native and cobalt-substituted enzymes

Summary The interaction of Zn(II)- and Co(II)-carbonic anhydrase (CA) with a series of compounds possessing moieties resembling the aromatic sulfonamides, such as sulfamide, sulfamic acid, N -substituted aromatic sulfonamides, sulfenamides, sulfinic and seleninic acids, was investigated using kinetic and spectroscopic techniques. All these compounds inhibit the hydrasic and esterasic activity of the enzyme. Their binding within the active site of isozymes I and II is discussed on the basis of modifications of electronic and 1 H-NMR spectra of their adducts with the Co(II) enzyme. Some of these compounds represent novel classes of CA inhibitors, possessing equal or stronger potencies than the prototypical inhibitors, the unsubstituted sulfonamides. Qualitative structure-activity correlations are discussed.

pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins.

The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.

Fast acquisition of multi-dimensional spectra in solid-state NMR enabled by ultra-fast MAS

The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored. It is shown that optimal conditions for performing solid-state (13)C NMR under 60 kHz MAS are obtained with low-power CW (1)H decoupling, as well as after a low-power (1)H,(13)C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet-van Vleck theory. As a result, optimal (13)C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short (1)H T(1)s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D (13)C-(13)C correlation could be acquired in about 24 h on less than 1 mg of SOD sample.

Band-Selective 1H−13C Cross-Polarization in Fast Magic Angle Spinning Solid-State NMR Spectroscopy

A magic angle spinning (MAS) NMR technique to transfer polarization from protons to a specific set of the (13)C spins is introduced for the study of biomolecular samples in the solid-state. Ultrafast (>60 kHz) MAS and low irradiation rf fields are used to achieve band-selective Hartmann-Hahn cross-polarization (CP) between the whole proton bath and carbons whose resonances are close to the (13)C-transmitter offset. When compared to conventional, broadband (1)H-(13)C CP, the band-selective experiment can be established without any loss of sensitivity when polarizing the aliphatic signals of a protein sample, and with a significant gain when polarizing carbonyls. This scheme can be used as a building block in 2D (13)C-(13)C homonuclear correlation experiments to obtain a faster and more sensitive characterization of biological solids.

Magic Angle Spinning NMR of Paramagnetic Proteins

Metal ions are ubiquitous in biochemical and cellular processes. Since many metal ions are paramagnetic due to the presence of unpaired electrons, paramagnetic molecules are an important class of targets for research in structural biology and related fields. Today, NMR spectroscopy plays a central role in the investigation of the structure and chemical properties of paramagnetic metalloproteins, linking the observed paramagnetic phenomena directly to electronic and molecular structure. A major step forward in the study of proteins by solid-state NMR came with the advent of ultrafast magic angle spinning (MAS) and the ability to use (1)H detection. Combined, these techniques have allowed investigators to observe nuclei that previously were invisible in highly paramagnetic metalloproteins. In addition, these techniques have enabled quantitative site-specific measurement of a variety of long-range paramagnetic effects. Instead of limiting solid-state NMR studies of biological systems, paramagnetism provides an information-rich phenomenon that can be exploited in these studies. This Account emphasizes state-of-the-art methods and applications of solid-state NMR in paramagnetic systems in biological chemistry. In particular, we discuss the use of ultrafast MAS and (1)H-detection in perdeuterated paramagnetic metalloproteins. Current methodology allows us to determine the structure and dynamics of metalloenzymes, and, as an example, we describe solid-state NMR studies of microcrystalline superoxide dismutase, a 32 kDa dimer. Data were acquired with remarkably short times, and these experiments required only a few milligrams of sample.

Speeding Up 13C Direct Detection Biomolecular NMR Spectroscopy

After the exploitation of (1)H polarization as a starting source for (13)C direct detection experiments, pulse sequences are designed which exploit the accelerated (1)H longitudinal relaxation to expedite (13)C direct detection experiments. We show here that 2D experiments based on (13)C direct detection on a 0.5 mM water sample of ubiquitin can be recorded in a few minutes and 3D experiments in a few hours. We also show that fast methods like nonuniform sampling can be easily implemented. As overall experimental time has always been a counter indication for the use of (13)C direct detection experiments, this research opens new avenues for the application of (13)C NMR to biological molecules.