Automated Projection Spectroscopy Research Articles

Protein NMR Resonance Assignment without Spectral Analysis: 5D SOlid‐State Automated Projection SpectroscopY (SO‐APSY)

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible for the solid-state NMR analysis of proteins. We present the first ultrahigh dimensional implementation of this approach, where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular sizes and aggregation states, which show limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis and yield rapid and unbiased assignments of backbone resonances.

Automated projection spectroscopy in solid-state NMR.

Given that solid-state NMR is being used for protein samples of increasing molecular weight and complexity, higher-dimensionality methods are likely to be more and more indispensable for unambiguous chemical shift assignments in the near future. In addition, solid-state NMR spectral properties are increasingly comparable with solution NMR, allowing adaptation of more sophisticated solution NMR strategies for the solid state in addition to the conventional methodology. Assessing first principles, here we demonstrate the application of automated projection spectroscopy for a micro-crystalline protein in the solid state.

NMR structure determination of the protein NP_344798.1 as the first representative of Pfam PF06042.

We present the NMR structure determination of the protein {type:entrez-protein,attrs:{text:NP_344798.1,term_id:15900194,term_text:NP_344798.1}}NP_344798.1, which forms a CCA-adding enzyme head-domain architecture and is the first structural representative of the Pfam protein family PF06042. Its structure can now serve as a template for homology modeling of the other 785 members of this protein family. With 191 residues, {type:entrez-protein,attrs:{text:NP_344798.1,term_id:15900194,term_text:NP_344798.1}}NP_344798.1 is the largest single-domain protein structure determined so far with the J-UNIO protocol for automated NMR structure determination. The present work thus also shows that J-UNIO based exclusively on automated projection spectroscopy (APSY) and 3D heteronuclear-resolved [1H,1H]-NOESY experiments, can successfully be used to obtain high-quality NMR structures of protein domains with up to 200 residues.

A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins.

Sequence specific resonance assignment is the prerequisite for the NMR-based analysis of the conformational ensembles and their underlying dynamics of intrinsically disordered proteins. However, rapid solvent exchange in intrinsically disordered proteins often complicates assignment strategies based on HN-detection. Here we present a six-dimensional alpha proton detection-based automated projection spectroscopy (APSY) experiment for backbone assignment of intrinsically disordered proteins. The 6D HCACONCAH APSY correlates the six different chemical shifts, H(α)(i - 1), C(α)(i - 1), C'(i - 1), N(i), Cα(i) and Hα(i). Application to two intrinsically disordered proteins, 140-residue α-synuclein and a 352-residue isoform of Tau, demonstrates that the chemical shift information provided by the 6D HCACONCAH APSY allows efficient backbone resonance assignment of intrinsically disordered proteins.

Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10.

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80% of the resonances of the (13)C-(1)H moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.

4D experiments measured with APSY for automated backbone resonance assignments of large proteins

Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40 kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a 'just-in-time' manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90 % of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4 kDa.

Automated Projection Spectroscopy (APSY) for the Assignment of NMR Resonances of Biological Macromolecules

High resolution nuclear magnetic resonance (NMR) spectroscopy in solution is an established technique in structural biology. Detailed functional and structural studies of biological macromolecules by NMR require the assignment of the chemical shifts to specific nuclei. In biological applications, the necessary data is usually obtained from a number of two- and three-dimensional (2D and 3D) NMR experiments. Often, these data cannot be fully analyzed by automated computer programs due to insufficient separation and resolution of the signals in the available spectra. Then, complete resonance assignment requires manual interaction and can become a long and labor-intensive task. Automated projection spectroscopy (APSY) allows the substantial improvement of the resolution by providing spectral information from four and higher dimensional experiments without measuring the full spectrum, which would by far exceed any acceptable measuring time. APSY only measures a series of projections of the high-dimensional spectrum which can be obtained in a much shorter time. Peak picking of the projection spectra provides the basis for the calculation of the high-dimensional chemical shift correlation space by the algorithm GAPRO. The resulting high-dimensional peak lists are commonly artifact-free and of exceptional precision. Along with their high number of correlated nuclei they provide an ideal basis for reliable automated assignment. We will introduce the basic concepts of APSY, illustrate them with an application of a 6D APSY-seq-HNCOCANH experiment, and discuss some practical aspects.

Sugar-to-base correlation in nucleic acids with a 5D APSY-HCNCH or two 3D APSY-HCN experiments

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all (15)N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.

4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on (1)H(β) and (13)C(δ) efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13 kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCH(aro) sequence are proposed.

Automated NMR Resonance Assignment of Large Proteins for Protein−Ligand Interaction Studies

The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such interactions. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments, including the new 4D APSY-HNCACB, and the use of fractionally deuterated protein samples. This labeling pattern increases the experimental sensitivity on the one hand, but it leads to peak multiplets on the other hand. The latter complications are however overcome by the geometric APSY analysis of the projection spectra. The three APSY experiments thus provide high precision chemical shift correlations of the backbone and side chain methyl groups, allowing a reliable and robust assignment of the protein by suitable algorithms. The present approach doubles the molecular size limit of APSY-based assignments to 25 kDa, thus providing the basis for efficient characterization of protein-ligand interactions at atomic resolution by NMR, such as structure-based drug design. We show the application to two human proteins with molecular weights of 15 and 22 kDa, respectively, at concentrations of 0.4 mM and discuss the general applicability to studies of protein-protein and protein-nucleic acid complexes.

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 13 min. 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 assignments.

Automated NMR Assignment of Protein Side Chain Resonances Using Automated Projection Spectroscopy (APSY)

This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.

Sequence-Specific Resonance Assignment of Soluble Nonglobular Proteins by 7D APSY-NMR Spectroscopy

Based on sequence-specific resonance assignments, NMR is the method of choice for obtaining atomic-resolution experimental data on soluble nonglobular proteins. So far, however, NMR assignment of unfolded polypeptides in solution has been a time-consuming task, mainly due to the small chemical shift dispersion, which has limited practical applications of the NMR approach. This paper presents an efficient, fully automated method for sequence-specific backbone and beta-carbon NMR assignment of soluble nonglobular proteins with sizes up to at least 150 residues. The procedure is based on new APSY (automated projection spectroscopy) experiments which benefit from the short effective rotational correlation times in soluble nonglobular polypeptides to record five- to seven-dimensional NMR data sets, which reliably resolves chemical shift degeneracies. Fully automated sequence-specific resonance assignments of the backbone nuclei and C(beta) are described for the uniformly (13)C,(15)N-labeled urea-denatured 148-residue outer membrane protein X (OmpX) from E. coli. The method is generally applicable to systems with similar spectroscopic properties as unfolded OmpX, and we anticipate that this paper may open the door for extensive atomic-resolution studies of chemical denaturant-unfolded proteins, as well as some classes of functional nonglobular polypeptides in solution.

Automated projection spectroscopy (APSY)

This work presents the automated projection spectroscopy (APSY) method for the recording of discrete sets of j projections from N-dimensional (N > or = 3) NMR experiments at operator-selected projection angles and automatic identification of the correlation cross peaks. The result from APSY is the fully automated generation of the complete or nearly complete peak list for the N-dimensional NMR spectrum from a geometric analysis of the j experimentally recorded, low-dimensional projections. In the present implementation of APSY, two-dimensional projections of the N-dimensional spectrum are recorded by using techniques developed for projection-reconstruction spectroscopy [Kupce,E.& Freeman, R. (2004) J. Am. Chem. Soc. 126, 6429-6440]. All projections are peak-picked with the available automated routine atnos. The previously undescribed algorithm GAPRO (geometric analysis of projections) uses vector algebra to identify subgroups of peaks in different projections that arise from the same resonance in the N-dimensional spectrum, and from these subgroups it calculates the peak positions in the N-dimensional frequency space. Unambiguous identification thus can be achieved for all cross peaks that are not overlapped with other peaks in at least one of the N dimensions. Because of the correlation between the positions of corresponding peaks in multiple projections, uncorrelated noise is efficiently suppressed, so that APSY should be quite widely applicable for correlation spectra of biological macromolecules, which have intrinsically low peak density in the N-dimensional spectral space.