Giacomo Parigi

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.

The synthesis and in vitro testing of a zinc-activated MRI contrast agent

Zinc(II) plays a vital role in normal cellular function as an essential component of numerous enzymes, transcription factors, and synaptic vesicles. While zinc can be linked to a variety of physiological processes, the mechanisms of its cellular actions are less discernible. Here, we have synthesized and tested a Zn(II)-activated magnetic resonance imaging (MRI) contrast agent in which the coordination geometry of the complex rearranges upon binding of Zn(II). In the absence of Zn(II) water is restricted from binding to a chelated Gd(III) ion by coordinating acetate arms resulting in a low relaxivity of 2.33 mM−1·s−1 at 60 MHz. Upon addition of Zn(II) the relaxivity of the Gd(III)–Zn(II) complex increases to 5.07 mM−1·s−1 and is consistent with one water molecule bound to Gd(III). These results were confirmed by nuclear magnetic relaxation dispersion analysis. There was no observed change in relaxivity of the Gd(III) complex when physiologically competing cations Ca(II) and Mg(II) were added. A competitive binding assay gave a dissociation constant of 2.38 × 10−4 M for the Gd(III)–Zn(II) complex. In vitro magnetic resonance images confirm that Zn(II) concentrations as low as 100 μM can be detected by using this contrast agent.

Solid-state NMR of proteins sedimented by ultracentrifugation

Relatively large proteins in solution, spun in NMR rotors for solid samples at typical ultracentrifugation speeds, sediment at the rotor wall. The sedimented proteins provide high-quality solid-state-like NMR spectra suitable for structural investigation. The proteins fully revert to the native solution state when spinning is stopped, allowing one to study them in both conditions. Transiently sedimented proteins can be considered a novel phase as far as NMR is concerned. NMR of transiently sedimented molecules under fast magic angle spinning has the advantage of overcoming protein size limitations of solution NMR without the need of sample crystallization/precipitation required by solid-state NMR.

Dynamic nuclear polarization at high magnetic fields in liquids.

MPI for Biophysical Chemistry Gottingen, Am Fassberg 11, 37077 Gottingen, Germany b Free University Berlin, Inst. of Experimental Physics, Arnimallee 14, 14195 Berlin, Germany Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy Bruker Biospin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany e Technische Universitat Munchen, Department of Chemistry, Lichtenbergstr. 4, 85747 Garching, Germany Goethe University Frankfurt, Max von Laue Strasse 7, 60438 Frankfurt, Germany

Field dependent dynamic nuclear polarization with radicals in aqueous solution.

The dynamic nuclear polarization (DNP) effect between the polarizers TEMPOL and trityl and the 1H protons of water solution was investigated at two different magnetic fields corresponding to electron pumping frequencies of 9 and 94 GHz. For TEMPOL, large DNP enhancements up to about −100 (9 GHz) and −20 (94 GHz) were observed by continuous microwave irradiation on one of the three nitroxide hyperfine lines. These enhancements are considerably larger than those ever reported for this system and also larger than the ones we obtained with the second proposed polarizer agent, the trityl radical, under the same conditions. 1H Overhauser coupling factors were extracted from NMRD relaxation dispersion experiments and used to estimate the degree of effective saturation of the TEMPOL EPR line. Our results show that nitroxide radicals are well-suited polarizers for DNP experiments in aqueous solutions at variable fields. The large enhancements open up attractive perspectives for the application potential of DNP in ...

NMR RELAXATION IN SOLUTION OF PARAMAGNETIC COMPLEXES: RECENT THEORETICAL PROGRESS FOR S ≥ 1

Publisher Summary This chapter focuses on the first place on the phenomenon of paramagnetic relaxation enhancement and mentions recent theoretical developments in the neighboring fields. The first issue that needs to be clarified is the relation between macroscopic, observable properties of nuclear spins, and their microscopic counterparts. In solutions of transition metal ions or complexes, one can commonly consider a situation where the ligands carrying nuclear spins can reside in two types of environment: in the coordination sphere of the paramagnetic metal ion or in the bulk. If the ligand contains only one type of magnetic nuclei or if interactions between nuclear spins can be disregarded, each of the two sites can be characterized by nuclear spin–lattice and spin–spin relaxation times, T 1 and T 2 , respectively.

Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed.

A Modular System for the Synthesis of Multiplexed Magnetic Resonance Probes

We have developed a modular architecture for preparing high-relaxivity multiplexed probes utilizing click chemistry. Our system incorporates azide bearing Gd(III) chelates and a trialkyne scaffold with a functional group for subsequent modification. In optimizing the relaxivity of this new complex, we undertook a study of the linker length between a chelate and the scaffold to determine its effect on relaxivity. The results show a strong dependence on flexibility between the individual chelates and the scaffold with decreasing linker length leading to significant increases in relaxivity. Nuclear magnetic resonance dispersion (NMRD) spectra were obtained to confirm a 10-fold increase in the rotational correlation time from 0.049 to 0.60 ns at 310 K. We have additionally obtained a crystal structure demonstrating that modification with an azide does not impact the coordination of the lanthanide. The resulting multinuclear center has a 500% increase in per Gd (or ionic) relaxivity at 1.41 T versus small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.

Accurate solution structures of proteins from X-ray data and a minimal set of NMR data: calmodulin-peptide complexes as examples.

A strategy for the accurate determination of protein solution structures starting from X-ray data and a minimal set of NMR data is proposed and successfully applied to two complexes of calmodulin (CaM) with target peptides not previously described. Its implementation in the present case is based on the use of lanthanide ions as substitutes for calcium in one of the four calcium binding sites of CaM and the collection of pseudocontact shift (pcs) and residual dipolar coupling (rdc) restraints induced by the paramagnetic metals. Starting from the crystal structures, new structural models are calculated that are in excellent agreement with the paramagnetic restraints and differ significantly from the starting crystal structures. In particular, in both complexes, a change in orientation of the first helix of the N-terminal CaM domain and of the whole C-terminal domain is observed. The simultaneous use of paramagnetic pcs and rdc restraints has the following crucial advantages: (i) it allows one to assess the possible presence of interdomain conformational freedom, which cannot be detected if the rdc values are derived from external orienting media; (ii) in the absence of significant conformational freedom, the global orientation tensor can be independently and precisely determined from pcs values, which are less sensitive than rdc values to the presence of local structural inaccuracies, and therefore (iii) the relative rearrangement of a domain or a secondary structure element with respect to the metal-bearing domain can be detected.

Nuclear spin relaxation in paramagnetic complexes of S=1: Electron spin relaxation effects

Electron spin relaxation for an S=1 system and its field dependence in the presence of static zero-field splitting (ZFS) has been described and incorporated in a model for nuclear spin-lattice relaxation in paramagnetic complexes in solution, proposed earlier by the group in Florence. Slow reorientation is assumed and the electron spin energy level structure (at any orientation of the molecule with respect to the laboratory frame) is described in terms of the Zeeman interaction and of the static ZFS. The electron spin relaxation is assumed to be caused by a transient ZFS modulated by the deformation of the complex described as a distortional (or pseudorotational) motion and the Redfield theory is used to derive the electron spin relaxation matrices. In the description of the electron spin relaxation we neglect any contribution from mechanisms involving modulation by reorientation, such as those of the static ZFS and the less important Zeeman interaction, as we limit ourselves to the slow-rotation limit (i...