Virus−Ligand Interactions:  Identification and Characterization of Ligand Binding by NMR Spectroscopy

Benie, Andrew J.; Moser, Rosita; Bäuml, Englbert; Blaas, Dieter; Peters, Thomas

doi:10.1021/ja027691e

Cited by 153 publications

(75 citation statements)

References 17 publications

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“…Most noteworthy, there is no size limit for the protein receptor and an assignment of protein resonances is not required. For example, this has rendered possible the investigation of ligands binding to liposome-integrated membrane proteins (27) or to native viruses (28). Until today, only the monofunctional UDP-GlcNAc 2-epimerase from Escherichia coli, which shows only 22% homology of the amino acid sequence to the mammalian bifunctional enzyme, has been characterized and crystallized (29).…”

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Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Blume

Benie

Stolz

et al. 2004

Journal of Biological Chemistry

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The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.

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Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Blume

Benie

Stolz

et al. 2004

Journal of Biological Chemistry

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“…A transferência de NOE intermolecular entre macromoléculas e moléculas pequenas foi originalmente observada e descrita há mais de 30 anos 10,11 , sendo aplicada na determinação estrutural de moléculas ligadas a receptores macromoleculares 12,13 . O NOE depende do tempo de correlação (τ c ) das moléculas ligadas e livres.…”

Section: O Efeito Overhauser Nuclear (Noe)unclassified

“…Essa alteração resulta da transferência de polarização entre núcleos acoplados dipolarmente via mecanismos de relaxação spin-rede (T 1 ). Estes experimentos têm a característica de "sondar" acoplamentos dipolares que são espectralmente "invisíveis" nas amostras líquidas, mas fornecem informações relevantes sobre os movimentos moleculares e distâncias intra e intermoleculares.A transferência de NOE intermolecular entre macromoléculas e moléculas pequenas foi originalmente observada e descrita há mais de 30 anos 10,11 , sendo aplicada na determinação estrutural de moléculas ligadas a receptores macromoleculares 12,13 . O NOE depende do tempo de correlação (τ c ) das moléculas ligadas e livres.…”

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Mapeamento das interações proteína-ligante através de técnicas de RMN de ¹H utilizando detecção do ligante

Figueiredo

Marsaioli

2007

Quím. Nova

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Recebido em 11/8/06; aceito em 16/2/07; publicado na web em 29/8/07 SCREENING PROTEIN-LIGAND INTERACTIONS USING 1 H NMR TECHNIQUES FOR DETECTING THE LIGAND. NMR is a valuable screening tool for the binding of ligands to proteins providing structural information on both protein and ligands and is thus largely applied to drug-discovery. Among the recent NMR techniques to probe weak binding protein-ligand complexes we have critically evaluated the advantages and disadvantages of STD (Saturation Transfer Difference), WaterLOGSY (Water Ligand Observation with Gradient Spectroscopy), NOE pumping and DOSY-NOESY (Diffusion-Ordered NOESY) using a mixture of BSA (bovine serum albumin) plus salicylic acid, caffeine, citric acid, adipic acid and D-glucose.Keywords: NMR; protein-ligand interactions; bovine serum albumin. INTRODUÇÃOOs fenômenos físico-químicos desencadeados pelas interações intermoleculares entre ligantes e receptores macromoleculares são a chave para o entendimento dos processos biológicos 1 . Neste contexto, a ressonância magnética nuclear (RMN) tornou-se a priori uma técnica poderosa no estudo de interações proteína-ligante, sendo capaz de detectar e quantificar as interações com alta sensibilidade sem o conhecimento prévio da função da proteína 2 . Entretanto, o monitoramento das interações através dos deslocamentos químicos das macromoléculas é difícil de ser analisado sem marcação isotópica. Isso faz com que o estudo das propriedades dos ligantes seja mais apreciado, pois, fornece espectros de RMN mais simplificados e resolvidos que os espectros das macromoléculas. Existem muitos métodos espectroscópicos para a observação do epitopo dos ligantes, como métodos baseados na relaxação 3 T 2 e transferência de NOE 4 . Entende-se por epitopo a superfície de contato macromolécula-ligante que pode ser mapeada através dos hidrogênios situados na interface. Os principais experimentos utilizados na obtenção do epitopo do ligante são o STD 5,6 ("Saturation Transfer Difference"), WaterLOGSY 7 ("Water Ligand Observation with Gradient Spectroscopy") e NOE pumping 8,9 . Já o experimento de DOSY-NOESY ("Diffusion-Ordered" NOESY) permite a obtenção do epitopo da proteína. Portanto, estas técnicas fornecem informações complementares acerca das interações proteína/ligante.Este trabalho está voltado para o estudo das funções de proteínas através de suas interações com metabólitos celulares e xenobióticos, aplicando-se técnicas de RMN 1 H e de transferência de NOE. O efeito overhauser nuclear (NOE)O NOE (Efeito Overhauser Nuclear) é definido como a mudança na área do sinal proveniente de um núcleo causada pela saturação do sinal de um segundo núcleo. Essa alteração resulta da transferência de polarização entre núcleos acoplados dipolarmente via mecanismos de relaxação spin-rede (T 1 ). Estes experimentos têm a característica de "sondar" acoplamentos dipolares que são espectralmente "invisíveis" nas amostras líquidas, mas fornecem informações relevantes sobre os movimentos moleculares e distâncias intra e intermoleculares.A t...

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“…strong binders might go undetected. The STD approach has also been successfully applied to study binding of small molecules to an integral membrane protein [322], virus particles [323], and living cells [324]. The STD technique is easily adapted for use in 2D NMR pulse schemes [321].…”

Section: Saturation Transfer Difference (Std) Spectroscopymentioning

confidence: 99%

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

Stangler¹,

Hartmann²,

Willbold³

et al. 2006

Zeitschrift für Physikalische Chemie

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Solution NMR / Structure / Dynamics / InteractionsHigh resolution liquid state nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for in vitro studies of structure and dynamics of soluble biological macromolecules under physiological conditions. The unique combination of atomically resolved structural data with both local and global dynamic features covering the entire range of time scales from picoseconds to seconds makes NMR the method of choice in a very diverse and rapidly growing array of biochemical, biomedical, and pharmaceutical applications. After briefly introducing the basic principles of liquid state NMR we review recent methodological and instrumental advances in the field of biologically focused high resolution NMR. The main emphasis of the second part is on molecular interactions. Such interactions are fundamental for the function of proteins in living systems, e.g. for signal transduction, enzymatic catalysis, and immune defense. The tremendous opportunities of high resolution NMR in the identification and detailed characterization of the sites and modes of molecular interactions will be demonstrated.

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Virus−Ligand Interactions: Identification and Characterization of Ligand Binding by NMR Spectroscopy

Cited by 153 publications

References 17 publications

Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Mapeamento das interações proteína-ligante através de técnicas de RMN de ¹H utilizando detecção do ligante

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

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