RESUMEN
We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by â¼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.
Asunto(s)
Antígenos del Grupo Sanguíneo de Lewis , Ramnosa , Enlace de Hidrógeno , Termodinámica , Polisacáridos , HidrógenoRESUMEN
Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.
Asunto(s)
Glicoproteínas/química , Manosa/química , Resonancia Magnética Nuclear Biomolecular , Ribonucleasas/química , GlicosilaciónRESUMEN
Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longer-lived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H2O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans' labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss "active" glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2-8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY's NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 µs molecular dynamics simulations.
RESUMEN
Multidimensional TOCSY and NOESY are central experiments in chemical and biophysical NMR. Limited efficiencies are an intrinsic downside of these methods, particularly when targeting labile sites. This study demonstrates that the decoherence imparted on these protons through solvent exchanges can, when suitably manipulated, lead to dramatic sensitivity gains per unit time in the acquisition of these experiments. To achieve this, a priori selected frequencies are encoded according to Hadamard recipes, while concurrently subject to looped selective inversion or selective saturation procedures. Suitable processing then leads to protein, oligosaccharide and nucleic acid cross-peak enhancements of ≈200-1000% per scan, in measurements that are ≈10-fold faster than conventional counterparts. The extent of these gains will depend on the solvent exchange and relaxation rates of the targeted sites; these gains also benefit considerably from the spectral resolution provided by ultrahigh fields, as corroborated by NMR experiments at 600 MHz and 1 GHz. The mechanisms underlying these experiments' enhanced efficiencies are analyzed on the basis of three-way polarization transfer interplays between the water, labile and non-labile protons, and the experimental results are rationalized using both analytical and numerical derivations. Limitations as well as further extensions of the proposed methods, are also discussed.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Ácidos Nucleicos/química , Polisacáridos/química , Proteínas/química , Sitios de Unión , Espectroscopía de Resonancia Magnética/estadística & datos numéricos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
As a direct consequence of technological advancements, the interest in direct detection of low-gamma/low-sensitivity heteronuclei for NMR experiments has been revived. Until recently, experimental development of 13C/15N detected experiments has been focused on protein NMR. In the present report, we extend the use of 13C-detected experiments to structural studies of glycans in natural abundance. The narrow 1H and wider 13C signal dispersion make glycans ideal candidates for heteronuclear detection. We show that 13C-detected HSQC offers a ten-fold increase in 13C dimension resolution compared to the analogous 1H-detected HSQC, when the experiments are acquired for the same amount of time. The enhanced resolution comes at the expense of 2 to 3-fold loss in SNR; however, the observed signal loss is a fraction of the theoretical 8-fold difference expected between experiments. Further, we show that by combining a 1H constant time element (CT), SMILE data reconstruction and 13C-direct detection, complete resonance assignments of highly degenerate glycan signals are possible. Finally, we demonstrate the potential of our strategy to aid in the assignment of complex glycans, by using a novel 13C-detected version of the CT-HSQC-TOCSY experiment performed on sialyl Lewis X pentasaccharide model system.
Asunto(s)
Isótopos de Carbono , Resonancia Magnética Nuclear Biomolecular/métodos , Polisacáridos/química , Algoritmos , Celobiosa , Hidrógeno/química , Oligosacáridos/química , Antígeno Sialil Lewis X/químicaRESUMEN
O-Acetylation of carbohydrates such as sialic acids is common in nature, but its role is not clearly understood due to the lability of O-acetyl groups. We demonstrated previously that 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) is a chemically and biologically stable mimic of the 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) of the corresponding sialoglycans. Here, a systematic nuclear magnetic resonance (NMR) spectroscopic and molecular dynamics (MD) simulation study was undertaken for Neu5,9Ac2-containing GM3 ganglioside glycan (GM3-glycan) and its Neu5Ac9NAc analog. GM3-glycan with Neu5Ac as the non-O-acetyl form of Neu5,9Ac2 was used as a control. Complete 1H and 13C NMR chemical shift assignments, three-bond 1H-13C trans-glycosidic coupling constants (3JCH), accurate 1H-1H coupling constants (3JHH), nuclear Overhauser effects and hydrogen bonding detection were carried out. Results show that structural modification (O- or N-acetylation) on the C-9 of Neu5Ac in GM3 glycan does not cause significant conformational changes on either its glycosidic dihedral angles or its secondary structure. All structural differences are confined to the Neu5Ac glycerol chain, and minor temperature-dependent changes are seen in the aglycone portion. We also used Density Functional Theory (DFT) quantum mechanical calculations to improve currently used 3JHH Karplus relations. Furthermore, OH chemical shifts were assigned at -10°C and no evidence of an intramolecular hydrogen bond was observed. The results provide additional evidence regarding structural similarities between sialosides containing 9-N-acetylated and 9-O-acetylated Neu5Ac and support the opportunity of using 9-N-acetylated Neu5Ac as a stable mimic to study the biochemical role of 9-O-acetylated Neu5Ac.
Asunto(s)
Teoría Funcional de la Densidad , Gangliósido G(M3)/química , Simulación de Dinámica Molecular , Polisacáridos/química , Ácidos Siálicos/química , Conformación de Carbohidratos , Gangliósido G(M3)/biosíntesis , Espectroscopía de Resonancia Magnética , Ácido N-Acetilneuramínico/química , Polisacáridos/biosíntesisRESUMEN
Corynebacterium diphtheriae, a Gram-positive, aerobic bacterium, is the causative agent of diphtheria and cutaneous infections. While mechanisms required for heme iron acquisition are well known in C. diphtheriae, systems involved in the acquisition of other metals such as zinc and manganese remain poorly characterized. In this study, we identified a genetic region that encodes an ABC-type transporter (iutBCD) and that is flanked by two genes (iutA and iutE) encoding putative substrate binding proteins of the cluster 9 family, a related group of transporters associated primarily with the import of Mn and Zn. We showed that IutA and IutE are both membrane proteins with comparable Mn and Zn binding abilities. We demonstrated that the iutABCD genes are cotranscribed and repressed in response to iron by the iron-responsive repressor DtxR. Transcription of iutE was positively regulated in response to iron availability in a DtxR-dependent manner and was repressed in response to Zn by the Zn-dependent repressor Zur. Electrophoretic mobility shift assays showed that DtxR does not bind to the iutE upstream region, which indicates that DtxR regulation of iutE is indirect and that other regulatory factors controlled by DtxR are likely responsible for the iron-responsive regulation. Analysis of the iutE promoter region identified a 50-bp sequence at the 3' end of the iutD gene that is required for the DtxR-dependent and iron-responsive activation of the iutE gene. These findings indicate that transcription of iutE is controlled by a complex mechanism that involves multiple regulatory factors whose activity is impacted by both Zn and Fe.IMPORTANCE Vaccination against diphtheria prevents toxin-related symptoms but does not inhibit bacterial colonization of the human host by the bacterium. Thus, Corynebacterium diphtheriae remains an important human pathogen that poses a significant health risk to unvaccinated individuals. The ability to acquire iron, zinc, and manganese is critical to the pathogenesis of many disease-causing organisms. Here, we describe a gene cluster in C. diphtheriae that encodes a metal importer that is homologous to broadly distributed metal transport systems, some with important roles in virulence in other bacterial pathogens. Two metal binding components of the gene cluster encode surface exposed proteins, and studies of such proteins may guide the development of second-generation vaccines for C. diphtheriae.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Corynebacterium diphtheriae/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Zinc/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas de la Membrana Bacteriana Externa/genética , Corynebacterium diphtheriae/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Familia de Multigenes , Regiones Promotoras GenéticasRESUMEN
Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates and to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds that will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study, we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer, followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete NMR peak assignment and detailed MS information on oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.
Asunto(s)
Glicoconjugados/análisis , Neisseria meningitidis/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/química , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Vacunas Conjugadas/análisis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa , Glicoconjugados/química , Glicopéptidos/análisis , Neisseria meningitidis/inmunología , Serogrupo , Toxoide Tetánico/análisis , Toxoide Tetánico/química , Vacunas Conjugadas/químicaRESUMEN
Over the years, structural characterizations of α(2-8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is ß, which has the [I]CO2- group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2- group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2-8)-polySia near the reducing end, as the ß configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2-). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-ßOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2-8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2-8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.
Asunto(s)
Ácidos Siálicos/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Conformación Molecular , Simulación de Dinámica Molecular , Oxidación-Reducción , Electricidad Estática , Estereoisomerismo , TermodinámicaRESUMEN
Hydrogen bonds (Hbonds) are important stabilizing forces in biomolecules. However, for glycans in aqueous solution, direct NMR detection of Hbonds is elusive because of their transient nature. Here, we present Isotope-based Natural-abundance TOtal correlation eXchange SpectroscopY (INTOXSY), a new 1H-13C heteronuclear single quantum coherence-total correlation spectroscopy based method, to extract OH groups' exchange rate constants (kex) for molecules in natural 13C abundance and show that OH Hbonds can be inferred from "slower" H/D kex. We evaluate kex measured with INTOXSY in light of those extracted with line-shape analysis. Subsequently, we use a set of common glycans to establish a kex reference basis set and to infer the existence of transient Hbonds involving OH donor groups. Then, we report kex values for a series of mono- and disaccharides, as well as for oligosaccharides sialyl Lewis X and ß-cyclodextrin, and compare the results with those from the reference set to extract Hbond information. Finally, we utilize NMR experimental data in conjunction with molecular dynamics simulations to establish donor and acceptor Hbond pairs. Our exchange rate measurements indicate that OH/OD exchange rates, kHD, values <10 s-1 are consistent with transient Hbond OH groups and potential acceptor groups can be uncovered through MD simulations.
Asunto(s)
Hidrógeno/química , Hidróxidos/química , Polisacáridos/química , Agua/química , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Soluciones/químicaRESUMEN
We describe the direct NMR detection of a C-H···O nonconventional hydrogen bond (Hbond) and provide experimental and theoretical evidence for conventional Hbonds in the pentasaccharide sialyl Lewis-X (sLe(X)-5) between 5 and 37 °C in water. Extensive NMR structural studies together with molecular dynamics simulations offer strong evidence for significant local dynamics in the Le(X) core and for previously undetected conventional Hbonds in rapid equilibrium that modulate structure. These NMR studies also showed temperature-dependent (1)H and (13)C line broadening. The resulting model emerging from this study is more complex than a simple rigid core description of Le(X)-like molecules and improves our understanding of stabilizing interactions in glycans.
Asunto(s)
Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oligosacáridos/química , Enlace de Hidrógeno , Antígeno Sialil Lewis XRESUMEN
We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.
Asunto(s)
Bacillus/metabolismo , Hexosas/química , Hexosas/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Bacillus/enzimología , Escherichia coli/genética , Fructosa/metabolismo , Fructosafosfatos/metabolismo , Hexosafosfatos/metabolismo , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Especificidad por SustratoRESUMEN
The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Polisacáridos/química , Animales , HumanosRESUMEN
Chemical exchange saturation transfer (CEST) is an NMR method that takes advantage of proton exchange between solute and solvent molecules in dynamic equilibrium, enabling the detection of the solute NMR signals with enhanced sensitivity. Herein, we report that the hydroxyl groups in a naturally occurring polysaccharide, α-2,8 polysialic acid in aqueous solution, yield very significant CEST effects even at 37°C where the resonances of the hydroxyl groups are not directly observed. We also report the assignments of the hydroxyl groups for the polymer and its oligomeric building blocks, from monomer to hexamer. We show that the same assignments can be made by either (1)H-(1)H TOCSY methods or (1)H-(13)C HSQC-TOCSY methods, to alleviate spectral overlap. Finally, we report the exchange rates of the OH groups with water and show how these rates can be used to select and fine-tune CEST effects.
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Hidróxidos/química , Espectroscopía de Resonancia Magnética/métodos , Ácidos Siálicos/químicaRESUMEN
We introduce the abundant hydroxyl groups of glycans as NMR handles and structural probes to expand the repertoire of tools for structure-function studies on glycans in solution. To this end, we present the facile detection and assignment of hydroxyl groups in a wide range of sample concentrations (0.5-1700 mM) and temperatures, ranging from -5 to 25 °C. We then exploit this information to directly detect hydrogen bonds, well-known for their importance in molecular structural determination through NMR. Via HSQC-TOCSY, we were able to determine the directionality of these hydrogen bonds in sucrose. Furthermore, by means of molecular dynamics simulations in conjunction with NMR, we establish that one out of the three detected hydrogen bonds arises from intermolecular interactions. This finding may shed light on glycan-glycan interactions and glycan recognition by proteins.
Asunto(s)
Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Polisacáridos/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Proteínas/química , Proteínas/metabolismo , Sacarosa/química , Temperatura , Trehalosa/químicaRESUMEN
Characteristic H-bonding patterns define secondary structure in proteins and nucleic acids. We show that similar patterns apply for α2-8 sialic acid (SiA) in H(2)O and that H-bonds define its structure. A (15)N,(13)C α2-8 SiA tetramer, (SiA)(4), was used as a model system for the polymer. At 263 K, we detected intra-residue through-H-bond J couplings between (15)N and C8 for residues R-I-R-III of the tetramer, indicating H-bonds between the (15)N's and the O8's of these residues. Additional J couplings between the (15)N's and C2's of the adjacent residues confirm the putative H-bonds. NH groups showing this long-range correlation also experience slower (1)H/(2)H exchange. Additionally, detection of couplings between H7 and C2 for R-II and R-III implies that the conformations of the linkers between these residues are different than in the monomers. These structural elements are consistent with two left-handed helical models: 2 residues/turn (2(4) helix) and 4 residues/turn (1(4) helix). To discriminate between models, we resorted to (1)H,(1)H NOEs. The 2(4) helical model is in better agreement with the experimental data. We provide direct evidence of H-bonding for (SiA)(4) and show how H-bonds can be a determining factor for shaping its 3D structure.
Asunto(s)
Antígenos/química , Conformación de Carbohidratos , Ácido N-Acetilneuramínico/química , Oligosacáridos/química , Enlace de Hidrógeno , Espectroscopía de Resonancia MagnéticaRESUMEN
Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell-cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3-C2, H3-C1, and C1-C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the ~1% (13)C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.
Asunto(s)
Membrana Celular/química , Glicoproteínas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Polisacáridos/química , Ácidos Siálicos/química , Fragmentos Fc de Inmunoglobulinas/química , Ácido N-Acetilneuramínico/químicaRESUMEN
The ligand binding properties of the kringle 5 (K5) domain of human plasminogen have been investigated via intrinsic tryptophan fluorescence. The oleic acid (OA) affinity for K5 was quantified, yielding an association constant K(a) approximately 2.08 x 10(4) mM(-1). Simultaneously, it was determined that OA and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) (K(a) approximately 50 mM(-1)) compete for binding to K5. The solution structure of K5 in the presence of 11 mM AMCHA was solved via NMR spectroscopy (protein heavy atom RMSD approximately 0.93 +/- 0.12 A). The AMCHA binding site was localized via (1)H/(15)N chemical shift perturbation mapping assisted by in silico docking. We have found that AMCHA binds at the canonical kringle lysine binding site (LBS), structured by the Pro54-Gly60 segment plus the neighboring Phe36, Thr37, Trp62, Leu71, and Tyr72 residues. The segment 30-42, encompassing LBS residues, appears to be endowed with a higher degree of structural flexibility as suggested by the relatively lower value of S(2), the generalized order parameter, consistent with a higher backbone heavy atom RMSD of approximately 1.22 A (vs 0.84 A overall) between the two monomeric units in the crystal unit cell, of potential significance for ligand binding. OA was found to perturb the same area of the protein, namely, the LBS, as well as Tyr74. Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains.