Phase labeling of C−H and C−C spin-system topologies: Application in constant-time PFG-CBCA(CO)NH experiments for discriminating amino acid spin-system types

Summary Triple-resonance experiments facilitate the determination of sequence-specific resonance assignments of medium-sized ¹³C, ¹⁵N-enriched proteins. Some triple-resonance experiments can also be used to obtain information about amino acid spin-system topologies by proper delay tuning. The constant-time PFG-CBCA(CO)NH experiment allows discrimination between five different groups of amino acids by tuning (phase labeling) independently the delays for proton-carbon refocusing and carbon-carbon constant-time frequency labeling. The proton-carbon refocusing delay allows discrimination of spin-system topologies based on the number of protons attached to C and C atoms (i.e. C-H phase labeling). In addition, tuning of the carbon-carbon constant-time frequency-labeling delay discriminates topologies based on the number of carbons directly coupled to C and C atoms (i.e. C-C phase labeling). Classifying the spin systems into these five groups facilitates identification of amino acid types, making both manual and automated analysis of assignments easier. The use of this pair of optimally tuned PFG-CBCA(CO)NH experiments for distinguishing five spin-system topologies is demonstrated for the 124-residue bovine pancreatic ribonuclease A protein.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

Efficient side-chain and backbone assignment in large proteins: Application to tGCN5

In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast ¹³C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a ¹³ C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOCSY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.     

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.     

Determination of the rotational dynamics and pH dependence of the hydrogen exchange rates of the arginine guanidino group using NMR spectroscopy

Summary An improved version of the constant-time HSQC experiment is presented that gives uniform sensitivity over the complete ¹³C bandwidth in ¹³C−¹H correlation experiments without creating artifacts in the methyl and aromatic regions of the spectra. The improvement is achieved by replacing the refocussing ¹³C 180° pulse in the evolution time by a combination of a full-power (22 kHz) hyperbolic secant 180° pulse that inverts and refocusses the entire ¹³C window, immediately followed by a selective 180° pulse on the CO region. Further improvement in signal-to-noise in the aromatic and methyl regions, although less spectacular, is obtained by replacing the other two 180° ¹³C pulses in the INEPT parts of the pulse sequence by full-power hyperbolic secant pulses. Results of simulations and experimental data are presented that demonstrate the excellent performance of the hyperbolic secant pulse for broadband inversion and show that refocussing of transverse magnetization occurs over the same bandwidth, albeit with a ¹³C signal phase that depends quadratically on offset. A further modification, in which one of the selective pulses on the CO region is omitted, is also presented. Implications for other 2D and 3D experiments performed at high fields, where uniform ¹³C inversion and refocussing is desirable, are discussed.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

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.     

Automated sequencing of amino acid spin systems in proteins using multidimensional HCC(CO)NH-TOCSY spectroscopy and constraint propagation methods from artificial intelligence

Summary We have developed an automated approach for determining the sequential order of amino acid spin systems in small proteins. A key step in this procedure is the analysis of multidimensional HCC(CO)NH-TOCSY spectra that provide connections from the aliphatic resonances of residue i to the amide resonances of residue i+1. These data, combined with information about the amino acid spin systems, provide sufficient constraints to assign most proton and nitrogen resonances of small proteins. Constraint propagation methods progressively narrow the set of possible assignments of amino acid spin systems to sequence-specific positions in the process of NMR data analysis. The constraint satisfaction paradigm provides a framework in which the necessary constraint-based reasoning can be expressed, while an object-oriented representation structures and facilitates the extensive list processing and indexing involved in matching. A prototype expert system, AUTOASSIGN, provides correct and nearly complete resonance assignments with one real and 31 simulated 3D NMR data sets for a 72-amino acid domain, derived from the Protein A of Staphylococcus aureus, and with 31 simulated NMR data sets for the 50-amino acid human type- transforming growth factor.     

Conditions for C NMR detection of 2-hydroxyglutarate in tissue extracts from isocitrate dehydrogenase-mutated gliomas

¹³C NMR (nuclear magnetic resonance) spectroscopy of extracts from patient tumor samples provides rich information about metabolism. However, in isocitrate dehydrogenase (IDH)-mutant gliomas, ¹³C labeling is obscured in oncometabolite 2-hydroxyglutaric acid (2HG) by glutamate and glutamine, prompting development of a simple method to resolve the metabolites. J-coupled multiplets in 2HG were similar to glutamate and glutamine and could be clearly resolved at pH 6. A cryogenically cooled ¹³C probe, but not J-resolved heteronuclear single quantum coherence spectroscopy, significantly improved detection of 2HG. These methods enable the monitoring of ¹³C–¹³C spin–spin couplings in 2HG expressing IDH-mutant gliomas.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully¹³ C/¹⁵N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue¹³ C-¹⁵N correlation experiments with¹³ C-¹³C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using ¹⁵N instead of¹³ C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the ¹⁵N and ¹³C resonances in human ubiquitin.     

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.     

Sequential individual resonance assignments in the 1H-nmr spectra of polypeptides and proteins

Recently, a new procedure for the assignment of protein ¹H-nmr spectra was introduced that relies on stereochemical considerations of proton–proton distances in polypeptides and on the use of two-dimensional nmr for obtaining ¹H-¹H through-bond and through-space connectivity maps. In the present paper a particular aspect of this assignment procedure is discussed in more detail, i.e., how to obtain individual resonance assignments from identification of amino acid side-chain spin systems and identification of neighboring residues in the amino acid sequence.     

Improved lineshape and sensitivity in the HNCO-family of triple resonance experiments

A simple scheme is presented for the suppression of dispersive contributions to cross peaks in HNCO-type spectra where the ¹⁵N chemical shift is recorded in a constant-time manner immediately prior to the transfer from ¹⁵ N to ¹HN at the end of the sequence. These dispersive contributions arise when the delay for refocusing the ¹⁵N-¹³CO one-bond coupling is set to less than 0.5/¹J_N,CO and when ² J_HN,CO 0. Improvements in sensitivity in ¹HN detected experiments recorded on ¹⁵ N,¹³C-labeled samples can be realized by application of ¹³CO/¹³ decoupling during acquisition. Sensitivity gains on the order of 15% and 5% have been obtained for an SH3 domain (62 residues) and maltose binding protein (370 residues), respectively.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.