Jean-Philippe Demers

Binding Mechanism of an SH3 Domain Studied by NMR and ITC

Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, DeltaH(D)(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, DeltaS(D)(30 degrees C) = 20.0 cal mol(-1) K(-1). The free protein and peptide have significantly higher heat capacity than the bound complex, DeltaC(p) = 352 cal mol(-1) K(-1), which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, DeltaH(D)(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of DeltaC(p). The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K(D), and range from k(on)(10 degrees C) = 1.03 x 10(8) M(-1) s(-1) to k(on)(50 degrees C) = 2.0 x 10(8) M(-1) s(-1), with an apparent activation enthalpy, DeltaH(A)(double dagger) = 3.4 kcal mol(-1). Both the magnitudes and temperature dependence of k(on) values are consistent with a diffusion-limited association mechanism. The combination of NMR and ITC data sheds light on how the Fyn tyrosine kinase is activated by binding to proline-rich targets, and represents a powerful approach for characterizing transient protein/ligand interactions.

Lewis base mediated autoionization of GeCl2 and SnCl2.

Cationic and anionic species of heavier low-valent group 14 elements are intriguing targets in main group chemistry due to their synthetic potential and industrial applications. In the present study, we describe the synthesis of cationic (MCl(+)) and anionic (MCl(3)(-)) species of heavier low-valent group 14 elements of germanium(II) and tin(II) by using the substituted Schiff base 2,6-diacetylpyridinebis(2,6-diisopropylanil) as Lewis base (LB). Treatment of LB with 2 equiv of GeCl(2)·dioxane and SnCl(2) in toluene gives compounds [(LB)Ge(II)Cl](+)[Ge(II)Cl(3)](-) (1) and [(LB)Sn(II)Cl](+)[Sn(II)Cl(3)](-) (2), respectively, which possess each a low-valent cation and an anion. Compounds 1 and 2 are well characterized with various spectroscopic methods and single crystal X-ray structural analysis.

Preparation of RSn(I)–Sn(I)R with Two Unsymmetrically Coordinated Sn(I) Atoms and Subsequent Gentle Activation of P4

This article reports the reduction of [{2,6-iPr(2)C(6)H(3)NC(CH(3))}(2)C(6)H(3)SnCl] (1) with potassium graphite to afford a new distannyne [{2,6-iPr(2)C(6)H(3)NC(CH(3))}(2)C(6)H(3)Sn](2) (2) with a Sn-Sn bond. The most striking phenomenon of 2 is the presence of two differently coordinated Sn atoms (one is three-coordinated, the other is four-coordinated). The Sn-Sn bond length in 2 is 2.8981(9) Å, which is very close to that of a Sn-Sn single bond (2.97-3.06 Å). To elucidate the nature of the Sn-Sn bond, DFT calculation is carried out that shows there is no multiple bond character in 2. Furthermore, the reaction of 2 with white P(4) affords the tetraphosphabicylobutane derivative 3. This is the first example of gentle activation of white phosphorus by a compound with low valent Sn atoms. Note that, unlike 2, in 3 both Sn atoms are four-coordinated.

Low-power solid-state NMR experiments for resonance assignment under fast magic-angle spinning.

Solid-state NMR has evolved in the past decade into a powerful technique for the characterization of biomolecular structure and dynamics. Micro-crystalline globular proteins, amyloid fibrils, and membrane proteins can now be routinely studied using solid-state NMR techniques. This was made possible in part due to the development of 2D and 3D homonuclear and heteronuclear experiments that correlate C and N spins for resonance assignment as well as for obtaining longrange distance restraints in structure elucidation. Remarkable developments in magic-angle spinning (MAS) probe technology also contributed to this success. Now, a new generation of commercially available 1.3 mm probes can reach above 60 kHz of MAS. This allows for more efficient averaging of strong dipolar couplings, hence providing better resolution in highly crowded protein spectra. On the other hand, fast spinning reduces the effectiveness of many of the routinely used NMR experiments for obtaining resonance assignments. For example, at low MAS ( 15 kHz), C C correlations are often measured by proton-driven spin diffusion (PDSD). Under very fast MAS, efficient averaging of dipolar couplings renders PDSD experiments ineffective. Instead, selective dipolar recoupling of spins becomes necessary to allow for efficient polarization transfer. Herein, we introduce a complete set of low-power solidstate NMR experiments sufficient for protein resonance assignment under fast MAS (>60 kHz), including sequential N C correlation experiments. The low rf (radio frequency)-field requirements of our experiments prevent considerable heating of the sample, thus avoiding protein degradation and making this approach well-suited for the investigation of temperaturesensitive biomolecules. As an application, NCA, N(CO)CA, and C C correlation spectra were recorded at 60 kHz MAS on less than 1 mg of [C, N] isotope-labeled sample. We also demonstrate that our approach can be readily performed on protein samples in which the H T1 relaxation times are shortened by means of paramagnetic doping. Here, the reduced recycle delay enhances the sensitivity but requires the use of NMR sequences with low-power deposition, as described herein. Figure 1 presents the different pulse schemes that were used to obtain N C and C C correlations. At low MAS, the initial cross-polarization (CP) transfer from protons to low-g nuclei generally requires high power irradiation on both channels. In contrast, under fast MAS, efficient CP transfer is also possible at low rf fields. Our pulse schemes use second-order cross-polarization (SOCP) for the initial magnetization transfer. SOCP at the n=0 Hartman–Hahn condition relies on second-order crossterms between homonuclear and heteronuclear couplings. SOCP works efficiently at low rf fields if sufficient care is taken to avoid detrimental dipolar and/or CSA recoupling conditions at the used rf-field amplitudes. We employed rf fields of 9 kHz—well below all resonance conditions. SOCP is intrinsically band-selective as only weak rf fields are applied. The rf fields employed here are sufficient to excite all N protein backbone

Monomeric Sn(II) and Ge(II) hydrides supported by a tridentate pincer-based ligand

Herein we report the syntheses of terminal Sn(II) (3) and Ge(II) (4) hydrides from the corresponding chloride precursors [{2,6-iPr(2)C(6)H(3)NCMe}(2)C(6)H(3)MCl] (M = Sn (1), Ge (2)) using [K{B(sec-Bu)(3)}H] as a hydrogenating agent. Combination of steric shielding and intramolecular N → M interactions resulted in the protection of M(II)-H bonds.

Studies on the MxiH protein in T3SS needles using DNP-enhanced ssNMR spectroscopy.

Bacterial T3SS needles formed by the protein MxiH are studied using DNP-enhanced ssNMR spectroscopy at 14.1 T (600 MHz). This technique provides spectra of good resolution, allowing us to draw conclusions about the protein dynamics. With the obtained signal enhancement, samples of limited quantity now get within reach of ssNMR studies.

Tailored low-power cross-polarization under fast magic-angle spinning.

High static magnetic fields and very fast magic-angle spinning (MAS) promise to improve resolution and sensitivity of solid-state NMR experiments. The fast MAS regime has permitted the development of low-power cross-polarization schemes, such as second-order cross-polarization (SOCP), which prevent heat deposition in the sample. Those schemes are however limited in bandwidth, as weak radio-frequency (RF) fields only cover a small chemical shift range for rare nuclei (e.g. (13)C). Another consideration is that the efficiency of cross-polarization is very sensitive to magnetization decay that occurs during the spin-lock pulse on the abundant nuclei (e.g. (1)H). Having characterized this decay in glutamine at 60 kHz MAS, we propose two complementary strategies to tailor cross-polarization to desired spectral regions at low RF power. In the case of multiple sites with small chemical shift dispersion, a larger bandwidth for SOCP is obtained by slightly increasing the RF power while avoiding recoupling conditions that lead to fast spin-lock decay. In the case of two spectral regions with large chemical shift offset, an extension of the existing low-power schemes, called MOD-CP, is introduced. It consists of a spin-lock on (1)H and an amplitude-modulated spin-lock on the rare nucleus. The range of excited chemical shifts is assessed by experimental excitation profiles and numerical simulation of an I(2)S spin system. All SOCP-based schemes exhibit higher sensitivity than high-power CP schemes, as demonstrated on solid (glutamine) and semi-solid (hydrated, micro-crystalline ubiquitin) samples.

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.

A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme

The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-(13)C]Glucose ([2-(13)C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-(13)C]pyruvate produced mainly by glycolysis and [3-(13)C]/[1,3-(13)C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs (13)Cβ-(13)Cγ2 and (13)Cα-(13)Cγ1 in Val, and (13)Cγ-(13)Cδ2 and (13)Cβ-(13)Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.

Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.

Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.