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1.
J Magn Reson ; 337: 107177, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35290935

RESUMO

We introduce an alternative way for spin-state selection, RODA, which yields higher sensitivity for spin systems exhibiting a TROSY effect. With RODA, the TROSY component of a doublet is recorded twice using a double acquisition scheme. RODA works by simple addition of consecutive NMR signals, and does not require any special processing. Thus, this pulse sequence element can seamlessly be integrated into existing experiments. We demonstrate the broad applicability of RODA with several systems exhibiting a TROSY effect on 15N-1H, 19F-13C or 1H-13C moieties. Further, we show that virtual decoupling with increased sensitivity is possible in a single double acquisition experiment in situations as encountered with dissolution DNP.


Assuntos
Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular
2.
J Cell Biol ; 218(9): 3117-3133, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31315942

RESUMO

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell-cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gßγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell-cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress-triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell-cell fusion during mating.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Mecânico , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/genética , Proteína Quinase C/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
MAbs ; 11(1): 94-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30570405

RESUMO

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.


Assuntos
Anticorpos Monoclonais/química , Biofarmácia/normas , Laboratórios/normas , Espectroscopia de Ressonância Magnética/métodos , Humanos , Reprodutibilidade dos Testes
4.
J Am Chem Soc ; 140(1): 167-175, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29192773

RESUMO

The homotrimeric ligand tumor necrosis factor α (TNFα) is a key cytokine and immune regulator; however, when deregulated, it leads to several major chronic inflammatory diseases. Perturbation of the protein-protein interface has proven to be an efficient strategy to inactivate TNFα, but the atomic-resolution mechanism of its inactivation remains poorly understood. Here, we probe the solution structure and dynamics of active and inactive TNFα using NMR spectroscopy. The data reveal that TNFα undergoes motions on different time scales. Furthermore, by site-directed mutagenesis of residues at the trimerization interface and by targeting the interface with a low molecular weight inhibitor, we show that TNFα retains its overall structure and trimeric state. However, upon perturbation, TNFα exhibits increased conformational dynamics spanning from the trimerization interface to the regions mediating receptor binding. These findings provide novel insights into the inactivation mechanism of TNFα and the basis for strategies to target TNFα activity.

5.
Angew Chem Int Ed Engl ; 54(24): 7096-100, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-25924827

RESUMO

Posttranslational modifications (PTMs) are an integral part of the majority of proteins. The characterization of structure and function of PTMs can be very challenging especially for glycans. Existing methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications, the inability to identify linkage types and thus chemical structure. We present a direct, robust, and simple NMR spectroscopy method for the detection and identification of PTMs in proteins. No isotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approach is well suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.


Assuntos
Proteínas/química , Sequência de Carboidratos , Glicosilação , Ressonância Magnética Nuclear Biomolecular , Polissacarídeos/análise , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
6.
Bioinformatics ; 31(18): 2981-8, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25995228

RESUMO

MOTIVATION: A detailed analysis of multidimensional NMR spectra of macromolecules requires the identification of individual resonances (peaks). This task can be tedious and time-consuming and often requires support by experienced users. Automated peak picking algorithms were introduced more than 25 years ago, but there are still major deficiencies/flaws that often prevent complete and error free peak picking of biological macromolecule spectra. The major challenges of automated peak picking algorithms is both the distinction of artifacts from real peaks particularly from those with irregular shapes and also picking peaks in spectral regions with overlapping resonances which are very hard to resolve by existing computer algorithms. In both of these cases a visual inspection approach could be more effective than a 'blind' algorithm. RESULTS: We present a novel approach using computer vision (CV) methodology which could be better adapted to the problem of peak recognition. After suitable 'training' we successfully applied the CV algorithm to spectra of medium-sized soluble proteins up to molecular weights of 26 kDa and to a 130 kDa complex of a tetrameric membrane protein in detergent micelles. Our CV approach outperforms commonly used programs. With suitable training datasets the application of the presented method can be extended to automated peak picking in multidimensional spectra of nucleic acids or carbohydrates and adapted to solid-state NMR spectra. AVAILABILITY AND IMPLEMENTATION: CV-Peak Picker is available upon request from the authors. CONTACT: gsw@mol.biol.ethz.ch; michal.walczak@mol.biol.ethz.ch; adam.gonczarek@pwr.edu.pl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Reconhecimento Visual de Modelos , Proteínas/química , Humanos
7.
Biochemistry ; 54(13): 2205-13, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25774789

RESUMO

Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability.


Assuntos
Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo I/química , Proteínas de Saccharomyces cerevisiae/química , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
J Biol Chem ; 289(50): 34953-64, 2014 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-25342741

RESUMO

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.


Assuntos
Agaricales/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Defensinas/farmacologia , Proteínas Fúngicas/farmacologia , Peptidoglicano/biossíntese , Agaricales/crescimento & desenvolvimento , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/crescimento & desenvolvimento , Técnicas de Cocultura , Defensinas/química , Defensinas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
9.
J Biomol NMR ; 59(4): 231-40, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24899400

RESUMO

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3 mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80 % of the resonances of the (13)C-(1)H moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.


Assuntos
Algoritmos , Automação/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , RNA Mensageiro/química , Animais , Proteínas de Drosophila , Drosophila melanogaster , Ressonância Magnética Nuclear Biomolecular/instrumentação , Proteínas Nucleares , Fatores de Transcrição
10.
J Biomol NMR ; 59(2): 87-93, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24771326

RESUMO

A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [(13)C, (15)N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1'C1'CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98 % assignment completeness for all (13)C-(1)H, (15)N1/9 and (31)P resonances of a stem-loop with 14 nucleotides.


Assuntos
Automação , Ressonância Magnética Nuclear Biomolecular , RNA/química
11.
J Mol Biol ; 426(3): 542-9, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24184277

RESUMO

Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation, where the incomplete Ig-like fold of FimA is completed by insertion of the N-terminal extension (donor strand) of the following FimA subunit. Recently, it was shown that FimA also exists in a monomeric, assembly-incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild-type FimA with its natural N-terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus, intramolecular self-complementation in the monomer stabilizes the FimA fold with significantly less interactions, and the natural FimA donor strand is inserted in the opposite orientation. In addition, we show that a motif of two glycine residues in the FimA donor strand, separated by five residues, is the prerequisite of the alternative, parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative, functionally relevant folding possibilities, with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells.


Assuntos
Escherichia coli/metabolismo , Proteínas de Fímbrias/química , Fímbrias Bacterianas/metabolismo , Teste de Complementação Genética , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Fímbrias/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas , Homologia de Sequência de Aminoácidos
12.
Angew Chem Int Ed Engl ; 53(5): 1320-3, 2014 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-24356903

RESUMO

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the ß and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M). Furthermore, it exhibits high thermal stability in native conditions. Binding of the Ste5 RING-H2 domain to the physiological Gß/γ (Ste4/Ste18) ligand is accompanied by a conformational transition into a better folded, more globular structure. This study reveals novel insights into the folding mechanism and recruitment of binding partners by the Ste5 RING-H2 domain. We speculate that many RING domains may share a similar mechanism of substrate recognition and molten-globule-like character.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Mercaptoetanol/química , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cloreto de Sódio/química , Temperatura , Ureia/química
13.
J Biomol NMR ; 56(2): 149-54, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23625454

RESUMO

Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40 kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a 'just-in-time' manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90 % of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4 kDa.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Peso Molecular , Ressonância Magnética Nuclear Biomolecular/métodos
14.
Chimia (Aarau) ; 66(10): 770-4, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23146263

RESUMO

High resolution nuclear magnetic resonance (NMR) spectroscopy in solution is an established technique in structural biology. Detailed functional and structural studies of biological macromolecules by NMR require the assignment of the chemical shifts to specific nuclei. In biological applications, the necessary data is usually obtained from a number of two- and three-dimensional (2D and 3D) NMR experiments. Often, these data cannot be fully analyzed by automated computer programs due to insufficient separation and resolution of the signals in the available spectra. Then, complete resonance assignment requires manual interaction and can become a long and labor-intensive task. Automated projection spectroscopy (APSY) allows the substantial improvement of the resolution by providing spectral information from four and higher dimensional experiments without measuring the full spectrum, which would by far exceed any acceptable measuring time. APSY only measures a series of projections of the high-dimensional spectrum which can be obtained in a much shorter time. Peak picking of the projection spectra provides the basis for the calculation of the high-dimensional chemical shift correlation space by the algorithm GAPRO. The resulting high-dimensional peak lists are commonly artifact-free and of exceptional precision. Along with their high number of correlated nuclei they provide an ideal basis for reliable automated assignment. We will introduce the basic concepts of APSY, illustrate them with an application of a 6D APSY-seq-HNCOCANH experiment, and discuss some practical aspects.


Assuntos
Substâncias Macromoleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteínas/química , Estereoisomerismo
15.
Top Curr Chem ; 316: 21-47, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21710379

RESUMO

This chapter presents the NMR technique APSY (automated projection spectroscopy) and its applications for sequence-specific resonance assignments of proteins. The result of an APSY experiment is a list of chemical shift correlations for an N-dimensional NMR spectrum (N≥3). This list is obtained in a fully automated way by the dedicated algorithm GAPRO (geometric analysis of projections) from a geometric analysis of experimentally recorded, low-dimensional projections. Because the positions of corresponding peaks in multiple projections are correlated, thermal noise and other uncorrelated artifacts are efficiently suppressed. We describe the theoretical background of the APSY method and discuss technical aspects that guide its optimal use. Further, applications of APSY-NMR spectroscopy for fully automated sequence-specific backbone and side chain assignments of proteins are described. We discuss the choice of suitable experiments for this purpose and show several examples. APSY is of particular interest for the assignment of soluble unfolded proteins, which is a time-consuming task by conventional means. With this class of proteins, APSY-NMR experiments with up to seven dimensions have been recorded. Sequence-specific assignments of protein side chains in turn are obtained from a 5D TOCSY-APSY-NMR experiment.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Software , Algoritmos , Automação , Padrões de Referência
16.
J Biomol NMR ; 52(2): 141-50, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22143941

RESUMO

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5 h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all (15)N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , RNA/química , Sequências Repetidas Invertidas , Ribose/química
17.
J Biomol NMR ; 51(3): 313-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21947871

RESUMO

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on (1)H(ß) and (13)C(δ) efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13 kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCH(aro) sequence are proposed.


Assuntos
Aminoácidos Aromáticos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono , Peso Molecular , Conformação Proteica , Prótons
18.
J Mol Biol ; 412(3): 520-35, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21816158

RESUMO

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (-10.1 kJ mol(-1)) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (-85.0 kJ mol(-1)) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive ß-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.


Assuntos
Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Dobramento de Proteína , Escherichia coli Uropatogênica/química , Proteínas de Escherichia coli , Cinética , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
19.
J Biol Chem ; 286(2): 1364-73, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-20929865

RESUMO

Ubiquitin-binding domains (UBDs) provide specificity to the ubiquitin system, which is also involved in translesion synthesis (TLS) in eukaryotic cells. Upon DNA damage, the UBDs (UBM domains) of polymerase iota (Pol ι) interact with ubiquitinated proliferating cell nuclear antigen to regulate the interchange between processive DNA polymerases and TLS. We report a biophysical analysis and solution structures of the two conserved UBM domains located in the C-terminal tail of murine Pol ι in complex with ubiquitin. The 35-amino acid core folds into a helix-turn-helix motif, which belongs to a novel domain fold. Similar to other UBDs, UBMs bind to ubiquitin on the hydrophobic surface delineated by Leu-8, Ile-44, and Val-70, however, slightly shifted toward the C terminus. In addition, UBMs also use electrostatic interactions to stabilize binding. NMR and fluorescence spectroscopy measurements revealed that UBMs bind monoubiquitin, and Lys-63- but not Lys-48-linked chains. Importantly, these biophysical data are supported by functional studies. Indeed, yeast cells expressing ubiquitin mutants specifically defective for UBM binding are viable but sensitive to DNA damaging conditions that require TLS for repair.


Assuntos
Dano ao DNA/fisiologia , DNA Polimerase Dirigida por DNA , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Animais , Sítios de Ligação/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , DNA Polimerase iota
20.
Biomol NMR Assign ; 3(1): 133-6, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19636964

RESUMO

Acyl carrier proteins participate in the synthesis of fatty acids. Here we report the NMR resonances assignment of the acyl carrier protein domain of the Saccharomyces cerevisiae fatty acid synthase which corresponds to the fragment 138A-302L in the primary structure. The assignment will allow performing NMR studies with the aim to investigate the intrinsic dynamics of this protein, and to study the structural changes upon apo-holo transformation in order to unveil the mechanism of binding of the growing acyl chain.


Assuntos
Proteína de Transporte de Acila/química , Ácido Graxo Sintases/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Isótopos de Carbono/química , Dados de Sequência Molecular , Isótopos de Nitrogênio/química , Estrutura Terciária de Proteína , Subunidades Proteicas , Prótons
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