Automated probabilistic method for assigning backbone resonances of (13C,15N)-labeled proteins

We present a computer algorithm for the automated assignment of polypeptide backbone and13C resonances of a protein of known primary sequence. Input to the algorithm consistsof cross peaks from several 3D NMR experiments: HNCA, HN(CA)CO, HN(CA)HA,HNCACB, COCAH, HCA(CO)N, HNCO, HN(CO)CA, HN(COCA)HA, and CBCA(CO)NH.Data from these experiments performed on glutamine-binding protein are analyzed statisticallyusing Bayes' theorem to yield objective probability scoring functions for matching chemicalshifts. Such scoring is used in the first stage of the algorithm to combine cross peaks fromthe first five experiments to form intraresidue segments of chemical shifts{Ni,HiN,Ci,Ci,Ci}, while the latter five are combined into interresiduesegments {Ci,Ci,Ci,Ni+1,HNi+1}. Given a tentative assignment of segments,the second stage of the procedure calculates probability scores based on the likelihood ofmatching the chemical shifts of each segment with (i) overlapping segments; and (ii) chemicalshift distributions of the underlying amino acid type (and secondary structure, if known). Thisjoint probability is maximized by rearranging segments using a simulated annealing program,optimized for efficiency. The automated assignment program was tested using CBCANH andCBCA(CO)NH cross peaks of the two previously assigned proteins, calmodulin and CheA.The agreement between the results of our method and the published assignments wasexcellent. Our algorithm was also applied to the observed cross peaks of glutamine-bindingprotein of Escherichia coli, yielding an assignment in excellent agreement with that obtainedby time-consuming, manual methods. The chemical shift assignment procedure described hereshould be most useful for NMR studies of large proteins, which are now feasible with the useof pulsed-field gradients and random partial deuteration of samples.     

1h, 13c, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli

1H, 13C, and 15N NMR assignments of the backbone atoms and -carbons have been madefor liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with226 amino acid residues and a molecular weight of 24,935 Da. GlnBP is a periplasmicbinding protein which plays an essential role in the active transport of L-glutamine throughthe cytoplasmic membrane. The assignments have been obtained from three-dimensionaltriple-resonance NMR experiments on a 13C,15N uniformly labeled sample as well asspecifically labeled samples. Results from the 3D triple-resonance experiments, HNCO,HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are themain sources used to make the resonance assignments. Other 3D experiments, such asHNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirmthe resonance assignments and to extend connections where resonance peaks are missing insome of the experiments mentioned above. We have assigned more than 95% of thepolypeptide backbone resonances of GlnBP. The result of the standard manual assignment isin agreement with that predicted by an automated probabilistic method developed in ourlaboratory. A solution secondary structure of the GlnBP–Gln complex has beenproposed based on chemical shift deviations from random coil values. Eight -helices and10 -strands are derived using the Chemical Shift Index method.     

1H, 13C and 15N assignments and chemical shift-derived secondary structure of intestinal fatty acid-binding protein

Summary Sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments have been established for rat intestinal fatty acid-binding protein complexed with palmitate (15.4 kDa) at pH 7.2 and 37°C. The resonance assignment strategy involved the concerted use of seven 3D triple-resonance expriments (CC-TOCSY, HCCH-TOCSY, HNCO, HNCA, ¹⁵N-TOCSY-HMQC, HCACO and HCA(CO)N). A central feature of this strategy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The CC-TOCSY experiment provided the unambiguous links between the side-chain spin systems observed in HCCH-TOCSY and the backbone correlations observed in the other experiments. Assignments were established for 124 of the 131 residues, although 6 of the 124 had missing amide ¹H resonances, presumably due to rapid exchange with solvent under these experimental conditions. The assignment database was used to determine the solution secondary structure of the complex, based on chemical shift indices for the ¹H, ¹³C, ¹³C and ¹³CO atoms. Overall, the secondary structure agreed well with that determined by X-ray crystallography [Sacchettini et al. (1989) J. Mol. Biol., 208, 327–339], although minor differences were observed at the edges of secondary structure elements.     

1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Summary ¹H, ¹³C and ¹⁵N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, ¹⁵N TOCSY-HMQC and ¹⁵N NOESY-HMQC experiments provided the H chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C chemical shifts and TOCSY ladders. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D ¹⁵N NOESY-HMQC data and C secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper.     

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy

We report here the backbone 1HN, 15N, 13C, 13CO, and 1H NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13C, and 13CO resonances) were used to establishsequential connections. The connections using 13C resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded -sheet (four parallel strands, one antiparallelstrand) was also determined. -Sheet regions were identified by cross-stranddN and dNN connections and by strong intraresidue dN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor.     

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.     

13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

Assignment of amide proton signals by combined evaluation of HN,NN and HNCA MAS-NMR correlation spectra

In this paper, we present a strategy for the ¹H^N resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the -spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H^N-N-C correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D (¹H-¹⁵N) and 3D (¹H-¹⁵N-¹³C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone ¹⁵N resonances and as an aid in the amide proton assignment.     

Arginine side chain assignments in uniformly 15N-labeled proteins using the novel 2D HE(NE)HGHH experiment

A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain ¹H and ¹⁵N resonances inuniformly ¹⁵N-labeled proteins. Correlations between¹H, ¹Hand ¹H are established on the basis of³J(¹⁵N,¹H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with ¹H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly¹⁵N-labeled proteins of 7 and 23 kDa molecular weight.     

Side-chain conformation and dynamics in a solid peptide: CP-MAS NMR study of valine rotamers and methyl-group relaxation in fully 13C-labelled antamanide

The effect of the crystal lattice on the side-chain conformation andside-chain dynamics in peptides is investigated by solid-state NMR, using thecyclic decapeptide antamanide as an example. The study takes advantage of the¹³C assignment of the backbone and side chains based on theresolution-enhanced 2D spin-diffusion spectra by heteronuclear and homonucleardecoupling. The spectra even allow for a stereospecific assignment of the-carbons of the valine residue. It is found that the valine side chaincoexists in two static rotamer conformations which have not been observed byX-ray crystallography. In addition, remarkable effects of the crystal packingon the methyl-group rotation frequency are found from ¹³Crelaxation measurements.     

Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data

One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called Nasca (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), Nasca extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that Nasca assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by Nasca have backbone RMSD 0.8–1.5 Å from the reference structures determined by traditional NMR approaches.     

Application of multiple-quantum line narrowing with simultaneous 1H and 13C constant-time scalar-coupling evolution in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments

Many triple-resonance experiments make use of one-bond heteronuclear scalar couplings toestablish connectivities among backbone and/or side-chain nuclei. In medium-sized(15–30 kDa) proteins, short transverse relaxation times of C single-quantum stateslimit signal-to-noise (S/N) ratios. These relaxation properties can be improved usingheteronuclear multiple-quantum coherences (HMQCs) instead of heteronuclear single-quantumcoherences (HSQCs) in the pulse sequence design. In slowly tumbling macromolecules, theseHMQCs can exhibit significantly better transverse relaxation properties than HSQCs.However, HMQC-type experiments also exhibit resonance splittings due to multiple two- andthree-bond homo- and heteronuclear scalar couplings. We describe here a family of pulsed-field gradient (PFG) HMQC-type triple-resonance experiments using simultaneous 1H and13C constant-time (CT) periods to eliminate the t1 dependence of these scalar couplingeffects. These simultaneous CT PFG-(HA)CANH and PFG-(HA)CA(CO)NH HMQC-typeexperiments exhibit sharper resonance line widths and often have better S/N ratios than thecorresponding HSQC-type experiments. Results on proteins ranging in size from 6 to 30 kDashow average methine CH HMQC:HSQC enhancement factors of 1.10 ± 0.15, withabout 40% of the cross peaks exhibiting better S/N ratios in the simultaneous CT-HMQCversions compared with the HSQC versions.     

Sensitivity enhancement in (HCA)CONH experiments

A novel sensitivity-enhancement technique is proposed for experiments which correlate protein backbone resonances and start with magnetization from ¹³C-¹H groups. The technique is based on replenishing magnetization lost by dipole-CSA cross-correlated relaxation of the ¹³C spin with ¹³C steady state magnetization. The principle is demonstrated for the (HCA)CONH experiment, resulting in 1.6-fold sensitivity enhancement compared to the HN(CA)CO experiment. Furthermore, other versions of the (HCA)CONH experiment were evaluated, including a novel experiment with spin-locking of transverse ¹³C-¹H two-spin coherence, and a cross-correlation compensated (CA)CONH experiment which starts from ¹³C rather than ¹H magnetization.     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation

Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the H^N connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly ¹³C/¹⁵N enriched P13 domain of mannose permease (31 kDa).     

Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Phase labeling of C−H and C−C spin-system topologies: Application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins

Summary Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH straight-through triple-resonance experiments that allow rapid and almost complete assignments of backbone H, ¹³C, ¹⁵N and H^N resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly C and H resonances: (i) C–H phase discrimination by tuning of the refocusing period _{a_f}; (ii) C–C phase discrimination by tuning of the ¹³C constant-time evolution period 2T_c. For small proteins, C–C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C–H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing ¹³C-¹³C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.