A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

Summary A general approach for assigning the resonances of uniformly ¹⁵N- and ¹³C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The ¹H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D ¹⁵N-edited ¹H, ¹H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete ¹³C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the ¹H, ¹³C, and ¹⁵N resonances of the FK506 binding protein denatured in 6.3 M urea.     

Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain

Summary Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Ga14. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.     

NMR spectroscopy of peptides and proteins

High resolution nuclear magnetic resonance (NMR) spectroscopy is the only method available for determining the three-dimensional structures of peptides and proteins in solution at atomic resolution. This article deals with a range of practical considerations associated with such studies, including sample preparation, instrumental setup, one- and two-dimensional NMR methods, interpretation of spectral data, and structure calculations.     

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).     

Improved frequency resolution in multidimensional constant-time experiments by multidimensional Bayesian analysis

Summary The resolution of spectral frequencies in NMR data obtained from discrete Fourier transformation (DFT) along D constant-time dimensions can be improved significantly through extrapolation of the D-dimensional free induction decay (FID) by multidimensional Bayesian analysis. Starting from Bayesian probability theory for parameter estimation and model detection of one-dimensional time-domain data [Bretthorst, (1990) J. Magn. Reson., 88, 533–551; 552–570; 571–595], a theory for the D-dimensional case has been developed and implemented in an algorithm called BAMBAM (BAyesian Model Building Algorithm in Multidimensions). BAMBAM finds the most probable sinusoidal model to account for the systematic portion of any D-dimensional stationary FID. According to the parameters estimated by the algorithm, the FID is extrapolated in D dimensions prior to apodization and Fourier transformation. Multidimensional Bayesian analysis allows for the detection of signals not resolved by the DFT alone or even by sequential one-dimensional extrapolation from mirror-image linear prediction prior to the DFT. The procedure has been tested with a theoretical two-dimensional dataset and with four-dimensional HN(CO)CAHA (Kay et al. (1992) J. Magn. Reson., 98, 443–450) data from a small protein (8 kDa) where BAMBAM was applied to the ¹³C and H constant-time dimensions.To whom correspondence should be addressed.     

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.     

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively <Superscript>13</Superscript>C-labelled proteins

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-¹³C]- and [2-¹³C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [¹³C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in ¹³C-¹³C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved 1HN-detected triple resonance TROSY-based experiments

A pulse scheme resulting in improved sensitivity in TROSY-based 1HN-detected triple resonance experiments is presented. The approach minimizes relaxation losses which occur during the transfer of transverse magnetization from 15N to 1HN immediately prior to detection. The utility of the method is demonstrated on a complex of methyl protonated, highly deuterated maltose binding protein (MBP, 370 residues) and - cyclodextrin. Sensitivity gains relative to previous TROSY schemes of approximately 10 and 20% are noted in HNCO spectra of MBP recorded at 25 and 5°C, respectively, corresponding to molecular correlation times of 23 and 46 ns.     

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.     

Improved 4D NMR experiments for the assignment of backbone nuclei in13C/15N labelled proteins

Summary We recently proposed a novel 4D NMR strategy for the assignment of backbone nuclei in¹³C/¹⁵N-labelled proteins (Boucher et al., 1992). Intra-residue (and many sequential) assignments are obtained from a HCANNH experiment, whereas sequential assignments are based on a complementary HCA(CO)NNH experiment. We present here new constant time 4D HCANNH, HCA(CO)NNH and HNCAHA experiments that are more sensitive. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.     

Measurement of15N-13C J couplings in staphylococcal nuclease

Summary ¹⁵N-C and¹⁵N-C J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with¹⁵N and¹³C. It is found that the^IJ_C'N coupling is similar for -sheet, J=14.8 ± 0.5 and for -helix, J = 14.8 ± 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J=15.6 ± 0.5). On average,¹J_NC are smaller for -helical residues (J=9.6 ± 0.3 Hz) compared to -sheet (J=10.9 ± 0.8 Hz) and a substantial difference is observed for²J_NC in -helices (J=6.4 ± 0.4 Hz) and -sheets (J=8.3 ± 0.8 Hz).Dedicated to the memory of Professor V.F. Bystrov     

Sequential backbone assignment of uniformly 13C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment

Summary A new ¹H−¹³C−³¹P triple resonance experiment is described which allows unambigous sequential backbone assignment in ¹³C-labeled oligonucleotides via through-bond coherence transfer from ³¹P via ¹³C to ¹H. The approach employs INEPT to transfer coherence from ³¹P to ¹³C and homonuclear TOCSY to transfer the ¹³C coherence through the ribose ring, followed by ¹³C to ¹H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of ³¹P_i coherence via C4′_i and C4′_i-1, because of the relatively large J′_PC4 couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4′_i and C4′_i-1 coherences are subsequently transferred to, amongst others, H1′_i and H1′_i-1, respectively, leading to a 2D ¹H−³¹P spectrum which allows a sequential assignment in the ³¹P−¹H1′ region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a ¹³C-labeled RNA hairpin with the sequence 5′(GGGC-CAAA-GCCU)3′.     

Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques

Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

Covariance NMR in higher dimensions: application to 4D NOESY spectroscopy of proteins

Elucidation of high-resolution protein structures by NMR spectroscopy requires a large number of distance constraints that are derived from nuclear Overhauser effects between protons (NOEs). Due to the high level of spectral overlap encountered in 2D NMR spectra of proteins, the measurement of high quality distance constraints requires higher dimensional NMR experiments. Although four-dimensional Fourier transform (FT) NMR experiments can provide the necessary kind of spectral information, the associated measurement times are often prohibitively long. Covariance NMR spectroscopy yields 2D spectra that exhibit along the indirect frequency dimension the same high resolution as along the direct dimension using minimal measurement time. The generalization of covariance NMR to 4D NMR spectroscopy presented here exploits the inherent symmetry of certain 4D NMR experiments and utilizes the trace metric between donor planes for the construction of a high-resolution spectral covariance matrix. The approach is demonstrated for a 4D (13)C-edited NOESY experiment of ubiquitin. The 4D covariance spectrum narrows the line-widths of peaks strongly broadened in the FT spectrum due to the necessarily short number of increments collected, and it resolves otherwise overlapped cross peaks allowing for an increase in the number of NOE assignments to be made from a given dataset. At the same time there is no significant decrease in the positive predictive value of observing a peak as compared to the corresponding 4D Fourier transform spectrum. These properties make the 4D covariance method a potentially valuable tool for the structure determination of larger proteins and for high-throughput applications in structural biology.     

A 4D HCCH-TOCSY experiment for assigning the side chain1H and13C resonances of proteins

Summary A 4D HCCH-TOCSY experiment is described for correlating and assigning the¹H and¹³C resonances of protein amino acid side chains that has several advantages over 3D versions of the experiment. In many cases, both the¹H and¹³C chemical shifts can be obtained in the 4D experiment from a simple inspection of the¹³C(3),¹H(4) planes extracted at the¹H(1)/¹³C(2) chemical shifts. Together with the 3D and 4D triple resonance experiments, this allows sequence-specific assignments to be obtained. In addition, the increased resolution of the 4D experiment compared to its 3D counterpart allows. automation of resonance assignments.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Computer-assisted assignment of 2D1H NMR spectra of proteins: Basic algorithms and application to phoratoxin B

Summary A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D¹H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich. G. Wider, G. Wagner and W. Braun (1982)J. Mol. Biol. 155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program calledCONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.