APSY-NMR with proteins: practical aspects and backbone assignment

Automated projection spectroscopy (APSY) is an NMR technique for the recording of discrete sets of projection spectra from higher-dimensional NMR experiments, with automatic identification of the multidimensional chemical shift correlations by the dedicated algorithm GAPRO. This paper presents technical details for optimizing the set-up and the analysis of APSY-NMR experiments with proteins. Since experience so far indicates that the sensitivity for signal detection may become the principal limiting factor for applications with larger proteins or more dilute samples, we performed an APSY-NMR experiment at the limit of sensitivity, and then investigated the effects of varying selected experimental parameters. To obtain the desired reference data, a 4D APSY-HNCOCA experiment with a 12-kDa protein was recorded in 13min. Based on the analysis of this data set and on general considerations, expressions for the sensitivity of APSY-NMR experiments have been generated to guide the selection of the projection angles, the calculation of the sweep widths, and the choice of other acquisition and processing parameters. In addition, a new peak picking routine and a new validation tool for the final result of the GAPRO spectral analysis are introduced. In continuation of previous reports on the use of APSY-NMR for sequence-specific resonance assignment of proteins, we present the results of a systematic search for suitable combinations of a minimal number of four- and five-dimensional APSY-NMR experiments that can provide the input for algorithms that generate automated protein backbone assignment

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional(6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighbor in gamidemoieties in proteins via the C′ and Cα nuclei, with efficient suppression of the back transfer from Cα to the originating amidemoiety. The automatic analysis of two-dimensional(2D) projections of this 6D experiment with the use of GAPRO (Hilleretal., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1-3) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small protein

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional(6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighbor in gamidemoieties in proteins via the C′ and Cα nuclei, with efficient suppression of the back transfer from Cα to the originating amidemoiety. The automatic analysis of two-dimensional(2D) projections of this 6D experiment with the use of GAPRO (Hilleretal., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1-3) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins

Cell-free protein synthesis of perdeuterated proteins for NMR studies

Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric for

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated "spin diffusion” due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experimen

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated ``spin diffusion`` due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations

Anatomy of the sign-problem in heavy-dense QCD

QCD at finite densities of heavy quarks is investigated using the density-of-states method. The phase factor expectation value of the quark determinant is calculated to unprecedented precision as a function of the chemical potential. Results are validated using those from a reweighting approach where the latter can produce a significant signalto-noise ratio. We confirm the particle–hole symmetry at low temperatures, find a strong sign problem at intermediate values of the chemical potential, and an inverse Silver Blaze feature for chemical potentials close to the onset value: here, the phase-quenched theory underestimates the density of the full theory