A pentacyclic reaction intermediate of riboflavin synthase

The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various 13C- and 15N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.

Reversible encapsulation by self-assembling resorcinarene subunits

Encapsulation complexes are assemblies in which a reversibly formed host more or less completely surrounds guest molecules. Host structures held together by hydrogen bonds have lifetimes in organic solvents of milliseconds to hours, long enough to directly observe the encapsulated guest by NMR spectroscopy. We describe here the action of alkyl ammonium compounds as guests that gather up to six molecules of the host module to form encapsulation complexes. The stoichiometry of the complexes—the largest hydrogen-bonded host capsules to date—is determined by the size and concentration of the guest.

Functional dynamics in the active site of the ribonuclease binase

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum kcat (1,500 s−1) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Acyl tunichlorins: a new class of nickel chlorins isolated from the Caribbean tunicate Trididemnum solidum.

A new class of nickel-containing chlorins (acyl tunichlorins) has been isolated from the Caribbean tunicate Trididemnum solidum. The structures of 28 of these nickel (II) hydroporphyrins were elucidated using mass spectrometry, one- and two-dimensional NMR spectroscopy, and chemical degradation/derivatization. Unique structural features of these compounds include the diversity of aliphatic side chains, which are derived from C14:0 to C22:6 fatty acids, and their location at an unprecedented position at C-2a on the hydroporphyrin nucleus. No chlorins with ester-linked acyl side chains at C-2a have been reported previously. Although the exact biological role that these compounds play in T. solidum remains unknown, acyl tunichlorins represent the only nickel-containing chlorins to be isolated from a living system and are the C-2a acyl derivatives of tunichlorin, a nickel chlorin reported by this laboratory in 1988.

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin.

A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524].

Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve.

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.

Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.

Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.

One sequence, two folds: a metastable structure of CD2.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.0-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Propriedades fotoquímicas de alguns complexos de ferro(II) e rênio(I)

Neste trabalho, foram estudadas as propriedades fotoquímica e/ou fotofísica de alguns compostos de coordenação de rênio(l) e ferro(I I). A irradiação dos complexos fac-[Re(CO)3(NN)(trans-L)]+, NN= 4,7-difenil-1,10- fenantrolina (ph2phen) ou 5-cloro-1,10-fenantrolina (Clphen) e L = 1,2-bis(4-piridil)etileno (bpe) ou 4-estirilpiridina (stpy), em acetonitrila ou em filme de poli(metacrilato de metila) (PMMA) resulta em variações espectrais condizentes com a fotoisomerização trans-cis do ligante coordenado. A determinação dos rendimentos quânticos para a fotorreação pela variação espectral resultou em valores aparentes, uma vez que o reagente e o fotoproduto absorvem na mesma região. Para a determinação do rendimento quântico real, Φreal, utilizou-se a técnica de 1H RMN, na qual os sinais do fotoproduto e do reagente são observados em regiões distintas com diferentes constantes de acoplamento. Os valores de Φreal obtidos para fac-[Re(CO)3h(NN)(trans-bpe)]+ (ph2phen: Φ313= 0,43 ± 0,03; Φ365= 0,44 ± 0,02; Φ404= 0,43 ± 0,02; Clphen: Φ313= 0,56 ± 0,03; Φ365= 0,55 ± 0,04; Φ404= 0,57 ± 0,06) são independentes do comprimento de onda de irradiação, indicando a existência de um único canal para a população do estado excitado 3ILtrans-bpe. Por outro lado, para fac-[Re(CO)3(NN)(trans-stpy)]+, os valores de Φreal sob irradiação a 404 nm são menores que os determinados para os demais comprimentos de onda de irradiação (ph2phen: Φ313= 0,60 ± 0,05; Φ365= 0,64 ± 0,09; Φ404= 0,42 ± 0,03; Clphen: Φ313= 0,52 ± 0,05; Φ365= 0,58 ± 0,02; Φ404= 0,41 ± 0,06), indicando que, a energias maiores, em que o Iigante absorve significativamente, deve existir a contribuição de outro canal para a população do estado excitado 3ILtrans-stpy. A eficiência do fotoprocesso foi avaliada por meio da substituição dos ligantes NN e/ou L, e a diferença nos valores de Φreal entre os complexos deve estar relacionada principalmente com as distintas eficiências de cruzamento intersistemas. o fotoprocesso altera as propriedades fotofísicas desses complexos. Os isômeros trans apresentam fraca ou nenhuma emissão a 298 K, enquanto os fotoprodutos, fac-[Re(CO)3(NN)(cis-L)]+, apresentam intensa luminescência dominada pelo estado excitado 3MLCTRe→NN, que é sensivel à rigidez do meio. A reatividade fotoquímica dos pentacianoferratos(II) [Fe(CN)5 (NN)]3-, NN= 2aminobenzilamina (aba), 2-aminobenzamida (ab), 2-(dimetilaminometil)-3-hidroxipiridina (dmampy), 2-aminometilpiridina (ampy), 2-aminoetilpiridina (aepy) ou 2-(2metilaminoetil) piridina (maepy), também foi investigada. A irradiação desses complexos resulta na fotossubstituição do CN-, a qual só pode ser detectada quando o ligante possui um segundo grupo coordenante nas proximidades da esfera de coordenação. Os rendimentos quânticos da fotossubstituição são dependentes do comprimento de onda de irradiação (Φ313= 0,13 ± 0,01; Φ334= 0,091 ± 0,001; Φ365= 0,056 ± 0,002; Φ404= 0,022 ± 0,002; Φ436= 0,015 ± 0,001, por exemplo, para NN = aba) e indicam a existência de canais distintos pelos quais a fotorreação ocorre ou as diferentes eficiências de cruzamento intersistema para a população do estado excitado reativo. A eficiência do fotoprocesso também depende do Iigante utilizado (λirr= 365 nm: Φaba= 0,056, Φab= 0,14, Φampy= 0,046, Φaepy= 0,066, Φmaepy= 0,069 e Φdmampy= 0,12). Na série das diaminas, o rendimento quântico é maior para [Fe(CN)5(ab)]3-, que possui dois sítios para ocorrer o fechamento do anel. Na série das aminopiridinas, observa-se a influência do comprimento da cadeia na eficiência do fechamento do anel. A presença de metilas ligadas ao nitrogênio alifático deve ter pouca ou nenhuma influência na eficiência do fotoprocesso.