Multiple-quantum HCN-CCH-TOCSY experiment for 13C/15N labeled RNA oligonucleotides

A multiple-quantum 3D HCN-CCH-TOCSY experiment is presented for the assignment of RNA ribose resonances. The experiment makes use of the chemical shift dispersion of N1 of pyrimidine and N9 of purine to distinguish the ribose spin systems. It provides an alternative approach for the assignment of ribose resonances to the currently used COSY- and TOCSY-type experiments in which either ¹³C or ¹H is utilized to distinguish the different spin systems. Compared to the single-quantum version, the sensitivity of the multiple-quantum HCN-CCH-TOCSY experiment is enhanced on average by a factor of 2 for a 23-mer RNA aptamer complexed with neomycin.     

Sensitivity optimized HCN and HCNCH experiments for 13C/15N labeled oligonucleotides

Triple resonance HCN and HCNCH experiments used in studies of ¹³C/¹⁵N labeled oligonucleotides include extended evolution periods (typically up to 100 ms) to allow coherence transfer through a complex heteronuclear spin network. Unfortunately, most of the magnetization is lost during the evolution due to fast spin–spin relaxation dominated by one-bond ¹H–¹³C dipolar interaction. As demonstrated recently, the sensitivity of the experiments can be dramatically improved by keeping the spin system in a state of proton–carbon multiple-quantum coherence, which is not affected by the strong dipolar coupling. However, the multiple-quantum coherence is very sensitive to homonuclear as well as long-range heteronuclear interactions. Unwanted magnetization transfer due to these interactions can reduce the sensitivity back to the level of a single-quantum experiment and, for some spin moieties, even eliminate the signal completely. In the present paper we show that a modified HCN scheme that refocuses the interfering coherences improves sensitivity routinely by a factor of 1.5 to 4 over a nonselective experiment. In addition, novel multiple-quantum 2D and 3D HCNCH experiments with substantially enhanced sensitivity are presented.     

3d Hcch-Cosy-Tocsy experiment for the assignment of ribose and amino acid side chains in 13C labeled RNA and protein

A new 3D HCCH-COSY-TOCSY experiment is presented for the assignment of RNA sugar and protein side chains. The experiment, which combines COSY and TOCSY units, is more powerful than the sum of individual HCCH-COSY and HCCH-TOCSY pulse sequences. The experiment was applied to a 13C, 15N-labeled 26 mer RNA complexed with the antibiotic tobramycin, and a 12 kDa 13C, 15N-labeled FKBP12 protein sample. The power of HCCH-COSY-TOCSY is demonstrated through complete spin system assignments of sugars in the 26 mer RNA sample, which could not be assigned using a combination of HCCH-COSY, HCCH-TOCSY and 13C-edited NOESY experiments.     

Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

Summary The advent of methods for preparing ¹⁵N- and ¹³C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the ¹³C label. It may therefore sometimes be desirable to prepare a relatively inexpensive ¹⁵N-labeled sample only. Here we show that the two-bond ¹H-¹⁵N HSQC experiment can be used on ¹⁵N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected ¹H-¹⁵N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.     

Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides

Summary New 2D and 3D ¹H-¹³C-¹⁵N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in ¹³C-and ¹⁵N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective ¹³C-refocusing and ¹⁵N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ¹H-¹⁵N and 3D¹H-¹³C-¹⁵N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.     

Transverse relaxation optimized 3D and 4D 15N/15N separated NOESY experiments of 15N labeled proteins

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both ¹³C and ¹⁵N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach ¹⁵N-¹³C₁ residual dipolar couplings across a hydrogen bond as small as 20 ± 2 Hz have been characterized. While such distances are on the order of 4.2 ± 0.2 Å, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer–monomer interface defined this junction and confirmed the previous model of this interface.     

Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules

Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in ¹³C,¹⁵N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10°C and 25°C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA.     

Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment

Summary A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in ¹³C-/¹⁵N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.     

Long-range imino proton-13C J-couplings and the through-bond correlation of imino and non-exchangeable protons in unlabeled DNA

Thanks to rather large (5–9 Hz) long-range imino proton-¹³C J-couplings, heteronuclear correlation experiments in H₂O provide unambiguous assignment of imino protons by intranucleotide through-bond connectivities to guanosine H8 and thymidine CH₃ protons, or sequence-specific assignment of non-exchangeable protons when the imino protons are identified independently. This method is demonstrated in the Dickerson dodecamer [d(CGCGAATTCGCG)]₂ and in a human telomeric fragment of 22 nucleotides.     

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.     

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.     

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.     

Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins

Two triple resonance experiments, HNN and HN(C)N, are presented which correlate H^N and ¹⁵N resonances sequentially along the polypeptide chain of a doubly (¹³C, ¹⁵N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C and C chemical shift dispersions.     

Sequential correlation of anomeric ribose protons and intervening phosphorus in RNA oligonucleotides by a 1H,13C,31P triple resonance experiment: HCP-CCH-TOCSY

Summary A three-dimensional ¹H,¹³C,³¹P triple resonance experiment, HCP-CCH-TOCSY, is presented which provides unambiguous through-bond correlation of all ¹H ribose protons on the 5′ and 3′ sides of the intervening phosphorus along the backbone bonding network in ¹³C-labeled RNA oligonucleotides. The correlation of the complete ribose spin system to the intervening phosphorus is obtained by adding a C,C-TOCSY coherence transfer step to the triple resonance HCP experiment. The C,C-TOCSY transfer step, which utilizes the large and relatively uniform ¹J(C,C) coupling constant (∼40 Hz for ribose carbons), efficiently correlates the phosphorus-coupled carbons observed in the HCP correlation experiment (i.e., C4′ and C5′ in the 5′ direction and C4′ and C3′ in the 3′ direction) to all other carbons in the ribose spin system. Of the additional correlations observed in the HCP-CCH-TOCSY, that to the relatively well-resolved anomeric H1′, C1′ resonance pairs provides the greatest gain in terms of facilitating assignment. The gain in spectral resolution afforded by chemical shift labeling with the anomeric resonances should provide a more robust pathway for sequential assignment over the intervening phosphorus in larger RNA oligonucleotides. The HCP-CCH-TOCSY experiment is demonstrated on a uniformly ¹³C,¹⁵N-labeled 19-nucleotide RNA stem-loop, derived from the antisense RNA I molecule found in the ColE1 plasmid replication control system.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

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.     

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The ¹H, ¹³C, and ¹⁵N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C.     

Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins

A novel variant of the 13C/15N 2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

The use of heteronuclear cross-polarization for backbone assignment of 2H-, 15N- and 13C-labeled proteins: A pulse scheme for triple-resonance 4D correlation of sequential amide protons and 15N

Summary A new four-dimensional pulse scheme is described for the main-chain assignment of proteins by means of the J connectivity of the amide proton and nitrogen resonances of adjacent residues. Since the new experiment, 4D CP-HN(COCA)NH, involves heteronuclear cross-polarization for magnetization transfer from ¹³C=O to ¹⁵N via ¹³C, a relatively strong WALTZ-16 decoupling rf field is applied to ¹³C during magnetization transfer. Consequently, ¹³C is effectively decoupled from its attached ²H in the case of deuterated proteins, in the absence of a decoupling rf field for ²H. This efficiently improves the sensitivity of the experiment through ¹³C line narrowing. The experiment was performed on a randomly 60% deuterated protein, and the sensitivity of the final 4D spectrum was found to be excellent.