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Anja Böckmann

Publications and source records attributed to Anja Böckmann.

15 recordsLinked to original sources
PubMed2007

Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy.

We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.

Bacterial Proteins↗
PubMed2006

The influence of nitrogen-15 proton-driven spin diffusion on the measurement of nitrogen-15 longitudinal relaxation times.

The effect of nitrogen-15 proton-driven spin diffusion on quantitative (15)N T(1) measurements in solid proteins is investigated, and the impact on the measurement of dynamic parameters is assessed. A simple model of exchange between neighboring nitrogens is used to reproduce the evolution of (15)N spin systems whose longitudinal relaxation rates and exchange rates are compatible with experimental measurements. We show that the induced error in the measured T(1) and its effect on the determination of dynamics parameters is likely to be less than the current experimental error. The use of deuterated protein samples is shown to have a small but sometimes visible effect, and may also considerably slow down or even suppress the exchange of magnetization due to spin diffusion.

Artifacts↗
PubMed2006

Observation of heteronuclear overhauser effects confirms the 15N-1H dipolar relaxation mechanism in a crystalline protein.

The observation of proton to nitrogen-15 heteronuclear Overhauser effects in the microcrystalline protein Crh is used to confirm that the principal mechanism of relaxation of amide nitrogens is due to the fluctuation of the N-H dipolar couplings caused by N-H bond dynamics. Our observations reveal the central role of water as the main source of proton magnetization, and we provide an analysis of the different pathways that could lead to the observed results.

Bacterial Proteins↗
PubMed2006

Investigation of dipolar-mediated water-protein interactions in microcrystalline Crh by solid-state NMR spectroscopy.

Water-protein interactions play a major role in protein folding, structure, and function, and solid-state NMR has recently been shown to be a powerful tool for the site-resolved observation of these interactions in solid proteins. In this article we report investigations on possible water-protein dipolar transfer mechanisms in the microcrystalline deuterated protein Crh by a set of solid-state NMR techniques. Double-quantum (DQ) filtered and edited heteronuclear correlation experiments are used to follow direct dipolar water-protein magnetization transfers. Experimental data reveal no evidence for "solid-like" water molecules, indicating that residence times of solvent molecules are shorter than required for DQ creation, typically a few hundred microseconds. An alternative magnetization pathway, intermolecular cross-relaxation via heteronuclear nuclear Overhauser effects (NOEs), is probed by saturation transfer experiments. The significant additional enhancements observed when irradiating at the water frequency can possibly be attributed to direct heteronuclear water-protein NOEs; however, a contribution from relayed magnetization transfer via chemical exchange or proton-proton dipolar mechanisms cannot be excluded.

Carrier Proteins↗
PubMed2005

Quantitative analysis of backbone dynamics in a crystalline protein from nitrogen-15 spin-lattice relaxation.

A detailed analysis of nitrogen-15 longitudinal relaxation times in microcrystalline proteins is presented. A theoretical model to quantitatively interpret relaxation times is developed in terms of motional amplitude and characteristic time scale. Different averaging schemes are examined in order to propose an analysis of relaxation curves that takes into account the specificity of MAS experiments. In particular, it is shown that magic angle spinning averages the relaxation rate experienced by a single spin over one rotor period, resulting in individual relaxation curves that are dependent on the orientation of their corresponding carousel with respect to the rotor axis. Powder averaging thus leads to a nonexponential behavior in the observed decay curves. We extract dynamic information from experimental decay curves, using a diffusion in a cone model. We apply this study to the analysis of spin-lattice relaxation rates of the microcrystalline protein Crh at two different fields and determine differential dynamic parameters for several residues in the protein.

Bacterial Proteins↗
PubMed2005

Proton to carbon-13 INEPT in solid-state NMR spectroscopy.

A refocused INEPT through-bond coherence transfer technique is demonstrated for NMR of rigid organic solids and is shown to provide a valuable building block for the development of NMR correlation experiments in biological solids. The use of efficient proton homonuclear dipolar decoupling in combination with a direct spectral optimization procedure provides minimization of the transverse dephasing of coherences and leads to very efficient through-bond (1)H-(13)C INEPT transfer for crystalline organic compounds. Application of this technique to 2D heteronuclear correlation spectroscopy leads to up to a factor of 3 increase in sensitivity for a carbon-13 enriched sample in comparison to standard through-bond experiments and provides excellent selectivity for one-bond transfer. The method is demonstrated on a microcrystalline sample of the protein Crh (2 x 10.4 kDa).

Journal Article↗
PubMed2005

Water-protein hydrogen exchange in the micro-crystalline protein crh as observed by solid state NMR spectroscopy.

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T2' -selective 1H-13C-13C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T2' selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on 13C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.

Amino Acid Sequence↗
PubMed2004

Probing molecular interfaces using 2D magic-angle-spinning NMR on protein mixtures with different uniform labeling.

A general NMR strategy to directly study molecular interfaces under magic angle spinning is introduced. The approach is based on the spectroscopic analysis of uniformly, but heterogeneously, labeled molecular mixtures containing the spin species X and Y (X:Y). For the case of an ((15)N:(13)C) labeled sample, the use of NC, NHC, and NHHC transfers is demonstrated. Applied to the ((13)C:(15)N) labeled dimeric form of the 85 amino acid protein Crh, the NHHC approach reveals a variety of monomer-monomer interactions in the microcrystalline state.

Amino Acid Sequence↗
PubMed2003

NMR spectra of a microcrystalline protein at 30 kHz MAS.

Proteins are not always available in amounts desirable for solid-state magic-angle spinning (MAS) nuclear-magnetic resonance (NMR) spectroscopy. To maximize the signal-to-noise ratio achievable with small samples, the filling factor must be optimized by using small-diameter MAS rotors. These rotors have the added benefit of allowing higher radio frequency field amplitudes during polarization transfer steps and during decoupling periods as well as allowing higher spinning frequencies. We demonstrate the advantages of relatively fast MAS (30 kHz using a 2.5 mm rotor) compared to MAS at 12 kHz for the 10.4 kDa model protein Crh with 93 residues and show that the signal-to-noise ratio in two-dimensional correlation spectra can be significantly improved by taking advantage of optimized pulse sequences available with rapid MAS.

Bacillus subtilis↗
PubMed2003

Resolution enhancement in multidimensional solid-state NMR spectroscopy of proteins using spin-state selection.

We show that the resolution of homonuclear multidimensional solid-state NMR correlation experiments can be significantly improved using transition selection and spin-state-selective polarization transfer techniques. The selection and transfer of single states allow the removal of the J-coupling contribution from the line width in both the direct and indirect spectral dimensions. This is demonstrated with a new spin-state-selective CO-Calpha correlation experiment, applied to a microcrystalline 85-residue protein. With this new sequence, all four components of the CO-Calpha cross-peaks are separated into different spectra, obtained by linear combination of four recorded data sets. Line narrowing of up to 44% was obtained on the protein sample for the spin-state-selective CO-Calpha spectrum compared to a standard spin-diffusion experiment. The new technique also allows an easy distinction between "direct" and "relayed" transfer cross-peaks.

Carbon Isotopes↗
PubMed2003

Dimerization of Crh by reversible 3D domain swapping induces structural adjustments to its monomeric homologue Hpr.

The crystal structure of the regulatory protein Crh from Bacillus subtilis was solved at 1.8A resolution and showed an intertwined dimer formed by N-terminal beta1-strand swapping of two monomers. Comparison with the monomeric NMR structure of Crh revealed a domain swap induced conformational rearrangement of the putative interaction site with the repressor CcpA. The resulting conformation closely resembles that observed for the monomeric Crh homologue HPr, indicating that the Crh dimer is the active form binding to CcpA. An analogous dimer of HPr can be constructed without domain swapping, suggesting that HPr may dimerize upon binding to CcpA. Our data suggest that reversible 3D domain swapping of Crh might be an efficient regulatory mechanism to modulate its activity.

Amino Acid Sequence↗
PubMed2003

Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh.

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

Amino Acid Sequence↗