RESUMO
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 â 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 â 3 cross-links are replaced by 3 â 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.
Assuntos
Apoproteínas/química , Bacillus subtilis/enzimologia , Imipenem/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptidil Transferases/química , Prótons , Acilação , Sequência de Aminoácidos , Isótopos de Carbono , Dados de Sequência Molecular , Isótopos de Nitrogênio , Estrutura Secundária de ProteínaRESUMO
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.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química , Teofilina/química , Isótopos de Carbono , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Prótons , RNA/metabolismo , Teofilina/metabolismo , TermodinâmicaRESUMO
The solution structure of the complex formed between d(CGATCG)(2) and 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol, a new antitumor drug under design, has been resolved using NMR spectroscopy and restrained molecular dynamic simulations. The drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the minor groove. Analysis of NMR data establishes a weak stacking interaction between the intercalated ligand and the DNA bases; however, the drug/DNA affinity is enhanced by a hydrogen bond between the hydroxyl group of the end of the intercalant side chain and the amide group of guanine G6. Unrestrained molecular dynamic simulations performed in a water box confirm the stability of the intercalation model. The structure of the intercalated complex enables insight into the structure-activity relationship, allowing rationalization of the design of new antineoplasic agents.
Assuntos
Antineoplásicos/química , DNA/química , Substâncias Intercalantes/química , Ácidos Nucleicos Heteroduplexes/química , Oligonucleotídeos/química , Purinas/química , Sítios de Ligação , Desoxirribose/química , Imidazóis/química , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , Organofosfatos/química , Prótons , Piridinas/química , Soluções , TermodinâmicaRESUMO
Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear 13C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any 13C-edited multidimensional NMR experiment. As an example we have chosen the 13C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , RNA/química , Sequência de Bases , Isótopos de Carbono , Hidrogênio/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência/químicaRESUMO
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.
Assuntos
Cianeto de Hidrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Sequência de Bases , Carboidratos/química , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Prótons , RNA/químicaAssuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Isótopos de Carbono , Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Hidrogênio , Proteínas de Membrana/química , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Conformação ProteicaRESUMO
The lipoate containing H-protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The transfer of the CH(2) group is catalyzed by the T-protein, which forms a 1:1 complex with the methylamine-loaded H-protein (Hmet). The methylamine group is then deaminated and transferred to the tetrahydrofolate-polyglutamate (H(4)FGlu(n)) cofactor of T-protein, forming methylenetetrahydrofolate-polyglutamate. The methylamine group is buried inside the protein structure and highly stable. Experimental data show that the H(4)FGlu(n) alone does not induce transfer of the methylene group, and molecular modeling also indicates that the reaction cannot take place without significant structural perturbations of the H-protein. We have, therefore, investigated the effect of the presence of the T-protein on the stability of Hmet. Addition of T-protein without H(4)FGlu(n) greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol(-1). Differences of the (1)H and (15)N chemical shifts of the H-protein in its isolated form and in the complex with the T-protein show that the interaction surface for the H-protein is localized on one side of the cleft where the lipoate arm is positioned. This suggests that the role of the T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.
Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Pisum sativum/enzimologia , Apoenzimas/metabolismo , Sítios de Ligação , Catálise , Coenzimas/metabolismo , Simulação por Computador , Estabilidade Enzimática , Formaldeído/metabolismo , Complexo Glicina Descarboxilase , Proteína H do Complexo Glicina Descarboxilase , Glicina Desidrogenase (Descarboxilante) , Hidrocarbonetos , Cinética , Metano/análogos & derivados , Metano/metabolismo , Metilaminas/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Soluções , Tetra-Hidrofolatos/metabolismo , Termodinâmica , Ácido Tióctico/metabolismoAssuntos
Aminoácido Oxirredutases/química , Proteínas de Transporte/química , Isoenzimas/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Animais , Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Bases de Dados Factuais , Escherichia coli/química , Complexo Glicina Descarboxilase , Proteína H do Complexo Glicina Descarboxilase , Glicina Desidrogenase (Descarboxilante) , Metilaminas/metabolismo , OxirreduçãoRESUMO
A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.
Assuntos
Sítios de Ligação , RNA Catalítico/química , Sequência de Bases , Relação Dose-Resposta a Droga , Íons , Magnésio/química , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatos/químicaRESUMO
Three pyrido[1,2-e]purines of increasing hydrophilicity have been synthesized to evaluate as anticancer agents. These drugs interact quite differently with a synthetic oligodeoxynucleotide d(CGATCG)2. [1] is very hydrophobic due to a phenyl residue in its side chain. It only shows limited interactions with the minihelix without any evidence of intercalation. [2] and [3], on the other hand, have one ([2]) or two ([3]) hydroxyl groups in their acyl chain and present rather amphiphilic properties. The result is a similar intercalation of these derivatives between C and G base pairs as revealed by intermolecular nOe, 1H and 31P chemical shift variations. Models for the intercalation of [2] are proposed using energy minimizations and molecular dynamics (MD) calculations subject to restraints from nOe connectivities. Simulations and experiments indicate weak stability and thus fast exchange of [2] in its intercalation site.
Assuntos
Antineoplásicos/síntese química , Oligonucleotídeos/metabolismo , Purinas/química , Purinas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo , Animais , Linhagem Celular , Substâncias Intercalantes/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Músculo Esquelético/metabolismo , Testes de Mutagenicidade , Conformação de Ácido Nucleico , Prótons , Purinas/síntese química , Sulfonamidas/síntese químicaRESUMO
The lipoate-dependent H protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The local structure and backbone dynamics of the methylamine-loaded H (Hmet), oxidized H (Hox), and H apoprotein (Hapo) have been investigated in solution. Filtered NOESY experiments using a [13C]Hmet as well as comparison of the heteronuclear shifts between the Hox and Hmet proteins demonstrate that the methylamine group is located inside a cleft of the protein. Furthermore, this group appears to be locked in this configuration as indicated by the high value of the activation energy (37 kcal/mol) of the global unloading reaction and by its restricted mobility, deduced from 13C relaxation measurements. Comparisons of the 1H and 15N chemical shifts and 15N relaxation in the three forms suggest that part of the lipoyl-lysine arm interacts with the protein polypeptide in the Hox and Hmet. The major change induced by the loading of the methylamine group concerns the C-terminal helix whose mobility becomes completely restricted compared to those of the Hox and Hapo. This C-terminal helix exhibits different reorientational characteristics in the three forms, which can be explained in the Hapo by a model consisting of a twisting motion about an axis passing through the helix. Our results indicate that the model of a freely swinging arm proposed for other lipoate-containing proteins is not acceptable in solution for the GDC. The implication of this observation in terms of the mechanism of the interaction of the H protein with the T protein, its physiological partner during the catalytic cycle, is discussed.
Assuntos
Aminoácido Oxirredutases/química , Mitocôndrias/enzimologia , Isótopos de Carbono , Complexo Glicina Descarboxilase , Proteína H do Complexo Glicina Descarboxilase , Glicina Desidrogenase (Descarboxilante) , Metilaminas/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Pisum sativum/enzimologia , Proteínas de Plantas/química , Conformação Proteica , Prótons , Temperatura , Termodinâmica , Ácido Tióctico/análogos & derivados , Ácido Tióctico/químicaRESUMO
The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.
Assuntos
Oligorribonucleotídeos/metabolismo , RNA Catalítico/metabolismo , Sítios de Ligação/genética , Guanosina/metabolismo , Inosina/metabolismo , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Prótons , RNA Catalítico/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
Assuntos
Broncodilatadores/química , Conformação de Ácido Nucleico , RNA/química , Teofilina/química , Cafeína/química , Citosina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , Sequência de Bases , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , RNA Catalítico/metabolismoRESUMO
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.
Assuntos
Grupo dos Citocromos c/química , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Isótopos de Carbono , Citocromos c2 , Heme/química , Hidrogênio , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Rhodobacter capsulatus/metabolismoRESUMO
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.
Assuntos
Guanosina/química , Conformação de Ácido Nucleico , RNA Catalítico/química , Sequência de Bases , Isótopos de Carbono , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Isótopos de Nitrogênio , PrótonsRESUMO
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.
Assuntos
Citidina/química , RNA/química , Uridina/química , Animais , Sequência de Bases , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , PrótonsRESUMO
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.
Assuntos
Grupo dos Citocromos c/química , Rhodobacter capsulatus/enzimologia , Amidas/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Magnetismo , Conformação ProteicaRESUMO
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.
Assuntos
Grupo dos Citocromos c/química , Espectroscopia de Ressonância Magnética , Rhodobacter capsulatus/química , Sequência de Aminoácidos , Substâncias Macromoleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
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.