Extensive 1H NMR resonance assignment of proteins using natural abundance gradient-enhanced 13C-1H correlation spectroscopy.

The reliability and completeness of 1H NMR resonance assignment can be improved by the use of 13C-1H HSQC correlation spectra on unlabelled protein samples using pulsed field gradients. This technique is illustrated on a 5.2 mM sample of the 79 residue Desulfovibrio vulgaris ferrocytochrome c553. Protons attached to the same carbon can be unambiguously paired in a HSQC spectrum. Contrary to 1H, most amino acids exhibit characteristic 13C chemical shift ranges, which can be used for 13C assignment. This technique is especially useful for long side chain residues, such as Gln, Glu, Lys, Arg.

Characterization of the dynamic properties of Rhodobacter capsulatus ferricytochrome c'--a 28 kDa paramagnetic heme protein.

The cytochrome c' are paramagnetic heme proteins generally consisting of two identical 14 kDa subunits. The recent assignment of the 1H and 15N resonances of the Rhodobacter capsulatus ferricytochrome c' has allowed characterization of the dynamic properties by measurement of the heteronuclear NOE for each resolved amide group. The relative importance of fast local motion and paramagnetic effect on nuclear relaxation were distinguished by comparison of the measured heteronuclear NOE with that of the overall experimental average. We show that the average experimental value of -0.16 corresponds to the rigid body motion expected for a spherical complex of 28 kDa. Residues 3-5, 50-55 and 69-70 exhibit decreased heteronuclear NOE due to local motions on a fast time scale with respect to molecular tumbling. Based on the X-ray crystal structure of the homologous cytochrome c' from Chromatium vinosum, the mobile regions correspond to the N-terminus of helix-1 and 2 regions of nonregular secondary structure located between helices-2 and -3.

1H and 13C NMR assignment and secondary structure of Chlorobium limicola f. thiosulfatophilum ferrocytochrome c555.

The 1H resonances of the ferrocytochrome c555 from the anaerobic green sulfur bacterium Chlorobium limicola f thio-sulfatophilum (strain Tassajara) have been assigned. Identification of spin systems and sequential assignment of 1H was accomplished by automated assignment computer programs followed by manual verification. In addition, 13C resonances have been extensively assigned by HSQC experiments at natural abundance. As determined by short-range NOE connectivities, 13C alpha chemical shifts, and HN exchange experiments, the secondary structure consists of 3 helices ranging from residues 3-13, 43-53 and 70-86. Interestingly, the second helix is significantly longer than observed by X-ray crystallography [1977, Proc. Natl. Acad. Sci. USA 74, 5244-5247]. A topological model of the cytochrome c555 is presented based on a small number of long-range NOE contacts. The helices are shown to pack onto the heme according to the pattern common to all class I cytochromes c.

Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects.

2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic 'loop-helix-beta-sheet' structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a 'horse saddle' topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.

Sequential 1H and 15N NMR resonance assignment and secondary structure of ferrocytochrome c2 from Rhodobacter sphaeroides.

Sequence-specific 1H and 15N assignments have been made for the amino acids of the ferrocytochrome c2 from Rhodobacter sphaeroides. Initial assignments were made by analysis of a series of homonuclear 2D COSY, TOCSY, and NOESY spectra obtained with the unlabeled protein. 2D and 3D 1H-15N correlated spectra obtained for a uniformly 15N-labeled ferrocytochrome c2 were used to confirm and extend the assignments. Partial 13C assignments have also been made by means of HSQC experiments on 13C at natural abundance, in particular for about two-thirds of the 13C alpha. Medium-range NOE connectivities, together with 3J(HC alpha NH) coupling constants, indicated the presence of five helices at positions 6-16, 60-68, 74-82, 84-91, and 109-120. No other regular secondary structure was observed. This folding is similar to that previously observed for the ferrocytochrome c2 of Rhodobacter capsulatus in solution, which exhibits approximately 50% sequence identity. Moreover, the rotation rates of the aromatic rings of phenylalanine or tyrosine, when conserved, were similar to those observed for R. capsulatus. Furthermore, C alpha H chemical shifts, which are sensitive to the secondary structure and ring current effects of the heme, appear to be very similar for the two proteins. Consequently, the solution structure of R. sphaeroides ferrocytochrome c2 appears to be very similar to that of R. capsulatus ferrocytochrome c2. These results are compared with the X-ray crystal structure of the R. sphaeroides ferrocytochrome c2.

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated L,D-transpeptidase from Bacillus subtilis.

The D,D-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel ß-lactam resistance mechanism involving L,D-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the L,D-transpeptidases. To date, only one class of the entire ß-lactam family, the carbapenems, is able to inhibit the L,D-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the L,D-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain (1)H, (15)N and (13)C NMR assignment of the L,D-transpeptidase from Bacillus subtilis (Ldt(Bs)) in the apo and in the acylated form with a carbapenem, the imipenem.

Transverse relaxation optimized HCN experiment for nucleic acids: combining the advantages of TROSY and MQ spin evolution.

A three-dimensional MQ-TROSY-HCN pulse sequence is presented which provides intra-base and sugar-to-base correlations for 13C, 15N labeled nucleic acids (RNA, DNA). The experiment simultaneously exploits the favorable relaxation properties of 1H-13C multiple quantum coherence for sugar carbons and of 13C TROSY-type spin evolution for base carbons. MQ-TROSY-HCN thus combines the advantages of MQ-HCN for sugar-to-base and TROSY-HCN for intra-base correlations in a single experiment. In addition, two slightly different implementations of the MQ-TROSY-HCN experiment ensure optimal performance for small and larger oligonucleotides, respectively. The advantages of the MQ-TROSY-HCN experiment compared to the best previous implementations of HCN are demonstrated for a 33 nucleotide RNA aptamer.

Computer assignment of the backbone resonances of labelled proteins using two-dimensional correlation experiments.

We present ALPS (Assignment for Labelled Protein Spectra), a flexible computer program for the automatic assignment of backbone NMR resonances of (15)N/(13)C-labelled proteins. The program constructs pseudoresidues from peak-picking lists of a set of two-dimensional triple resonance experiments and uses either a systematic search or a simulated annealing-based optimization to perform the assignment. This method has been successfully tested on two-dimensional triple resonance spectra of Rhodobacter capsulatus ferrocytochrome c (2) (116 amino acids).

A 2D NMR study of the internal flexibility of the antifungal peptide stendomycin.

A 2-D 1H NMR study (NOESY, COSY, HOHAHA and ROESY experiments) of the antifungal peptide stendomycin is presented. The variation of the NOESY cross peak intensities is measured as a function of temperature in order to discriminate between constant and fluctuating interproton distances. It is shown that among 71 NOESY cross peaks, only 12 correspond to well defined interproton distances and their correlation time is determined. The other cross peaks cannot be translated accurately in terms of distances owing to internal molecular motions. (1H)-13C nOe measurements confirm the internal mobility of the molecule. Finally a flexibility map of stendomycin can be established.

Refinement of local and long-range structural order in theophylline-binding RNA using (13)C-(1)H residual dipolar couplings and restrained molecular dynamics.

13C-(1)H residual dipolar couplings (RDC) have been measured for the bases and sugars in the theophylline-binding RNA aptamer, dissolved in filamentous phage medium, and used to investigate the long-range structural and dynamic behavior of the molecule in the solution state. The orientation dependent RDC provide additional restraints to further refine the overall structure of the RNA-theophylline complex, whose long-range order was poorly defined in the NOE-based structural ensemble. Structure refinement using RDC normally assumes that molecular alignment can be characterized by a single tensor and that the molecule is essentially rigid. To address the validity of this assumption for the complex of interest, we have analyzed distinct domains of the RNA molecule separately, so that local structure and alignment tensors experienced by each region are independently determined. Alignment tensors for the stem regions of the molecule were allowed to float freely during a restrained molecular dynamics structure refinement protocol and found to converge to similar magnitudes. During the second stage of the calculation, a single alignment tensor was thus applied for the whole molecule and an average molecular conformation satisfying all experimental data was determined. Semirigid-body molecular dynamics calculations were used to reorient the refined helical regions to a relative orientation consistent with this alignment tensor, allowing determination of the global conformation of the molecule. Simultaneously, the local structure of the theophylline-binding core of the molecule was refined under the influence of this common tensor. The final ensemble has an average pairwise root mean square deviation of 1.50 +/- 0.19 A taken over all heavy atoms, compared to 3.5 +/- 1.1 A for the ensemble determined without residual dipolar coupling. This study illustrates the importance of considering both the local and long-range nature of RDC when applying these restraints to structure refinements of nucleic acids.

NMR assignment of Rhodobacter capsulatus ferricytochrome c', a 28 kDa paramagnetic heme protein.

The cytochromes c' are paramagnetic heme proteins generally consisting of two identical 14 kDa subunits. The 1H and 15N resonances of the ferricytochrome c' from the purple phototrophic bacterium Rhodobacter capsulatus have been extensively assigned by the TOCSY-HSQC, NOESY-HSQC, HSQC-NOESY-HSQC, and HNHA 3D heteronuclear experiments performed on an 8 mM sample labeled with 15N. In addition, the 13C alpha and 13CO resonances were assigned by the HNCA and multiple-quantum HNCOCA 3D experiments performed on a 0.5 mM sample labeled with 13C and 15N. The assignment of the backbone 13C resonances was used to confirm the 1H and 15N assignments and to better define secondary structure. On the basis of medium-range NOEs, 3JHN alpha coupling constants, and backbone 13C chemical shifts, the secondary structure consists of four helices: helix-1 (3-29), helix-2 (33-49), helix-3 (78-97), and helix-4 (103-117). On the basis of long-range NOE contacts, the Rb. capsulatus ferricytochrome c' is a four-helix bundle protein in which consecutive helices are antiparallel with respect to one another.

High-resolution 3D HNCOCA experiment applied to a 28 kDa paramagnetic protein.

A new triple-resonance 3D HNCOCA pulse scheme is presented, designed to identify the backbone nuclei (H(N), N, CO, C(α)) of doubly labelled proteins. The two carbon frequencies are labelled along the same indirect dimension and the corresponding dwell times can be independently scaled in order to account for the relaxation properties and chemical shift ranges of the CO and C(α). If one takes advantage of the symmetry properties of the spectra in the course of the peak picking, this 3D scheme has the same sensitivity as the 4D experiment, but with an improved resolution. The sequence is illustrated on a 0.5 mM sample of Rhodobacter capsulatus cytochrome c' a homodimeric paramagnetic protein of 2×14 kDa. A resonance assignment strategy, based on a low-concentration (13)C/(15)N-labelled sample and a more concentrated (15)N-labelled sample, is proposed for proteins where the expression system shows a limited efficiency.

Assignment of NMR spectra of proteins using triple-resonance two-dimensional experiments.

Two-dimensional versions of HNCA and HNCO experiments are described, which provide essentially the same information as the 3D sequence. A multiple-quantum coherence involving either 15N and 13C alpha or 15N and 13CO is created. One of the two frequencies is given by the middle point between the two cross peaks (zero- and double-quantum) and the other by their separation. Quadrature detection can be performed on either nucleus, modifying only the appearance of the 2D spectrum, but not the information content. These experiments, named MQ-HNCA and MQ-HNCO, are illustrated on a (15N, 13C) doubly labelled cytochrome c2 from Rhodobacter capsulatus (116 amino acids).

Three-dimensional structure in solution of a wheat lipid-transfer protein from multidimensional 1H-NMR data. A new folding for lipid carriers.

Two-dimensional and three-dimensional 1H-NMR experimental data [Simorre, J. P., Caille, A., Marion, D., Marion, D. & Ptak, M. (1991) Biochemistry 30, 11600-11608] were used to build models of the three-dimensional structure of a non-specific wheat lipid-transfer protein (LTP) by using distance geometry, simulated annealing, energy minimization and molecular dynamics techniques. A first set of 881 distance constraints derived from NOE cross-peak intensities was used to generate 74 initial structures. One family of topological mirror images of the protein structure was eliminated by considering helical secondary-structure organization and steric requirements. Back calculations of NOE intensities led us to introduce 535 additional distance constraints. Finally, 21 structures were selected as representative of the structure of the protein. The polypeptide backbone folds into a simple and original right-handed winding. It is composed of a bundle of four helices linked by flexible loops, which is packed against a C-terminal fragment forming a non-standard saxophone-like shape. The folded protein is stabilized by hydrophobic interactions and the four disulfide bridges combined by pairs on each side of the protein. An hydrophobic cleft, formed by residues located in the second half of the protein could be a potential site for the binding of lipids.

Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA.

To visualize the interplay of RNA structural interactions in a ligand binding site, we have determined the solution structure of a high affinity RNA-theophylline complex using NMR spectroscopy. The structure provides insight into the ability of this in vitro selected RNA to discriminate theophylline from the structurally similar molecule caffeine. Numerous RNA structural motifs combine to form a well-ordered binding pocket where an intricate network of hydrogen bonds and stacking interactions lock the theophylline into the complex. Two internal loops interact to form the binding site which consists of a sandwich of three base triples. The complex also contains novel base-zipper and 1-3-2 stacking motifs, in addition to an adenosine platform and a reversed sugar. An important feature of the RNA is that many of the conserved core residues participate in multiple overlapping tertiary interactions. This complex illustrates how interlocking structural motifs can be assembled into a highly specific ligand-binding site that possesses high levels of affinity and molecular discrimination.

Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA.

A set of triple resonance experiments is presented, providing through-bond H2N/HN to H6 connectivities in uridines and cytidines in 13C-/15N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H2N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.

A conformational change in the catalytic core of the hammerhead ribozyme upon cleavage of an RNA substrate.

Heteronuclear multidimensional NMR structural studies have been performed on a hammerhead ribozyme complexed with a cleaved and an uncleaved substrate. The NMR data demonstrate that the three helices surrounding the conserved catalytic core hammerhead are stably formed in both complexes. Evidence is also presented that indicates that the sheared G-A base pairs in the conserved core are formed in the absence of Mg2+. The NMR structural data demonstrate that there is a significant structural change of the conserved core of the hammerhead ribozyme-substrate complex upon cleavage of the substrate. Molecular dynamics calculations were performed to generate models of the ribozyme-cleaved substrate complex, and these results are used to help understand the mechanism of the hammerhead cleavage reaction.

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

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.

Two- and three-dimensional 1H NMR studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure.

Two- and three-dimensional 1H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy proved to be a very efficient method for resonance assignments of protein 1H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by 3J alpha NH coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.

Assignment of the 13C and 13CO resonances for Rhodobacter capsulatus ferrocytochrome c2 using double-resonance and triple-resonance NMR spectroscopy.

Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two- and three-dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13C alpha and 13CO secondary shifts were better indicators of secondary structure than 1H alpha and 13C beta secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13C alpha and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.