Novel projected 4D triple resonance experiments for polypeptide backbone chemical shift assignment

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in ¹³C/¹⁵N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments, the ¹³C and ¹H chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularly useful for the assignment of glycine-rich polypeptide segments. Appropriate setting of the ¹H radiofrequency carrier allows one to place cross peaks correlating either backbone ¹⁵N/¹H^N/¹³C or ¹⁵N/¹H^N/¹H chemical shifts in separate spectral regions. Hence, peak overlap is not increased when compared with the conventional 3D HNNCA and HNN(CA)HA. 3D HNN[CAHA] and 3D HNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and HNN CACO, where ¹³C and ¹³C chemical shifts evolve in a common dimension. The ¹³C shift is detected in quadrature, which yields peak pairs encoding the ¹³C chemical shift in an in-phase splitting. This suite of four experiments promises to be of value for automated high-throughput NMR structure determination in structural genomics, where the requirement to independently sample many indirect dimensions in a large number of NMR experiments may prevent one from accurately adjusting NMR measurement times to spectrometer sensitivity.     

IBIS--a tool for automated sequential assignment of protein spectra from triple resonance experiments

We have developed a tool for computer-assisted assignments of protein NMR spectra from triple resonance data. The program is designed to resemble established manual assignment procedures as closely as possible. IBIS exports its results in XEASY format. Thus, using IBIS the operator has continuous visual and accounting control over the progress of the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential triple resonance connectivities within a few hours or days.     

Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments

In experiments with proteins of molecular weights around 100 kDa the implementation of [¹⁵N,¹H]-TROSY-elements in [¹⁵N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of sensitivity enhancement elements. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the ¹³ C ¹⁵N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the ¹⁵N multiplet components.     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

Sequence-specific NMR assignment of proteins by global fragment mapping with the program Mapper

A new program, Mapper, for semiautomatic sequence-specific NMR assignment in proteins is introduced. The program uses an input of short fragments of sequentially neighboring residues, which have been assembled based on sequential NMR connectivities and for which either the ¹³C and ¹³C chemical shifts or data on the amino acid type from other sources are known. Mapper then performs an exhaustive search for self-consistent simultaneous mappings of all these fragments onto the protein sequence. Compared to using only the individual mappings of the spectroscopically connected fragments, the global mapping adds a powerful new constraint, which results in resolving many otherwise intractable ambiguities. In an initial application, virtually complete sequence-specific assignments were obtained for a 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus.     

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.     

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 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.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

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′.     

(H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins

Triple resonance HN(COCA)NH pulse sequences for correlating 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins that utilize overlapping coherence transfer periods provide increased sensitivityrelative to pulse sequences that utilize sequential coherence transfer periods. Although theoverlapping sequence elements reduce the overall duration of the pulse sequences, theprincipal benefit derives from a reduction in the number of 180° pulses. Two versions of thetechnique are presented: a 3D (H)N(COCA)NH experiment that correlates 15N(i),1H(i-1), and 15N(i-1) spins, and a 3D HN(COCA)NH experiment that correlates 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins by simultaneously encoding the 1H(i) and 15N(i) chemical shiftsduring the t1 evolution period. The methods are demonstrated on a 13C/15N-enriched sampleof the protein ubiquitin and are easily adapted for application to 2H/13C/15N-enrichedproteins.     

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.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

CO_H(N)CACB experiments for assigningbackbone resonances in 13C/15N-labeledproteins

A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates¹H^N and ¹³CO spins with the¹³C and¹³C spins of adjacent amino acids. Thepulse sequence is an out-and-back design that starts with¹H^N magnetization and transfers coherence viathe ¹⁵N spin simultaneously to the ¹³CO and¹³C spins, followed by transfer to the¹³C spin. Two versions of the sequence arepresented: one in which the ¹³CO spins are frequency labeledduring an incremented t₁ evolution period prior to transfer ofmagnetization from the ¹³C to the¹³C resonances, and one in which the¹³CO spins are frequency labeled in a constant-time mannerduring the coherence transfer to and from the¹³C resonances. Because ¹³COand ¹⁵N chemical shifts are largely uncorrelated, thetechnique will be especially useful when degeneracy in the¹H^N-¹⁵N chemical shifts hindersresonance assignment. The CO_H(N)CACB experiment is demonstrated usinguniformly ¹³C/¹⁵N-labeled ubiquitin.     

[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13C evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110 kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time period.     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.     

Backbone and side-chain 1H, 13C, 15N resonance assignments of rat lipocalin2

Lipocalin2 plays an important role in the innate immune system. In this article we report the backbone and side-chain resonance assignments of rat lipocalin2 (rLcn2). These assignments provide a basis for determining the structure and dynamics of rLcn2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.