An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse

Ogura, Kenji; Terasawa, Hiroaki; Inagaki, Fuyuhiko

doi:10.1007/bf00228150

Cited by 99 publications

(102 citation statements)

References 27 publications

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“…The (Markley et al 1998 (Ogura et al 1996;Zwahlen et al 1997;Breeze 2000;Iwahara et al 2001) were recorded of the same sample to verify the assignment of IBS1 resonances (Supplemental Fig. S3).…”

Section: Nmr Spectroscopymentioning

confidence: 99%

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

Kruschel

Skilandat

Sigel

2014

RNA

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A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5 ′ exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5 ′ exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5 ′ exon and the intron. Here, we present the NMR solution structures of the d3 ′ hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg 2+ ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.

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Section: Nmr Spectroscopymentioning

confidence: 99%

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

Kruschel

Skilandat

Sigel

2014

RNA

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“…This was based on the design of Ogura et al (28), with the exception that hard 13 C 180° pulses were used instead of wide-band inversion pulses and that 90° 13 C pulses were added to generate double-quantum instead of antiphase coherence after the spin echoes. 2D f 1 -filtered 1H TOCSY and 1H NOESY (50 ms mixing time for both spectra) data were collected from the [U-13 C, U-15 N]-acyl-ACP samples.…”

Section: Nmr Of the Prosthetic Groupmentioning

confidence: 99%

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

2006

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Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo-and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0-and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0-and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners.Acyl carrier proteins participate in a wide variety of biosynthetic processes, including the synthesis of fatty acids (1), toxins (2), oligosaccharides (3,4), polyketides (5,6), biotin (7), and depsipeptides (8). They ferry small molecules between enzymes involved in biosynthesis by covalent linkage as a thioester to the thiol group of 4′-phosphopantetheine ( Figure 1). This prosthetic group is attached by a post-translational modification to a serine (S38 in spinach ACP 1 ) in the middle a conserved Asp-Ser-Leu (DSL) sequence. Whereas the sequences of different acyl carrier proteins are divergent, they share a similar four-helix bundle topology and often can be interchanged with full activity in reactions in vitro. This prompts the question: are ACPs simply passive molecules that constantly present their cargo to enzymes, or do protein-protein and protein-ligand interactions affect the utility of ACP as an enzyme substrate?One well-studied biosynthetic process involving ACP is the desaturation of the fatty acid stearate by the enzyme stearoyl-ACP Δ 9 -desaturase (Δ9D). This enzyme, in a reaction dependent on O 2 and reducing-equivalent, acts on ACP with an attached stearate (18:0-ACP) † This work was supported by the National Institutes of Health Grants R01 GM-50853 to B.G.F. and R01 GM-58667 to J.L.M. NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM), which is supported by grants from the NIH Center for Research Resources Biomedical Research Technology Program (P41 RR-02301) and the National Institutes of General Medical Sciences (P41 GM-GM66326) with additional instrumentation purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program, the NIH Shared Instrumentat...

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“…15 N/ 13 C-filtered 2D TOCSY (t m 21, 45 ms) and 2D NOESY (t m 50, 80, 100 and 150 ms) spectra were recorded using a double-tuned purging scheme as described by Ogura et al [31]. Water-flip back pulses were incorporated in filtered TOCSY experiments to remove antidiagonal spectral artifacts [32].…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

Sich¹,

Improta²,

Cowley³

et al. 2000

European Journal of Biochemistry

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The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein±ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15 N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.Keywords: FKBP12; ligand binding; nerve regeneration; NMR structure; protein dynamics.For several years there has been major interest in the development of inhibitors of the FK506 binding protein (FKBP12) because of their potential immunosuppressive effects [1±4]. Recently, FKBP12 was also identified as a target for the promotion of neurite outgrowth [5] and neurotrophic and neuroprotective effects could be associated with the action of different FKBP12 ligands [6,7]. These findings have extended the therapeutic potential of FKBP12-binding drugs to the treatment of nerve injuries and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.While it is well known that the immunosuppressive effects of FKBP12 are mediated by the interaction of the drug± immunophilin complex with calcineurin, the mechanism of the neuronal activity of FKBP12 has not yet been elucidated. However, it has been shown that the neurotrophic properties of immunophilin ligands are independent of their immunosuppressive action [8] and much effort has been directed toward the design of FKBP12-binding molecules, which are neurotrophic but nonimmunosuppressant, for clinical use [9,10]. One of the most promising compounds was discovered by scientists at Guilford Pharmaceuticals (1, Fig. 1; alternative name: GPI-1046). This ligand was synthesized within a series of small molecules that mimic the FKBP12-binding portion of FK506, a well-known immunosuppressant drug (Fig. 1), but lack the structural requirements for calcineurin inhibition. Among this series, 1 was found to be remarkably potent in inhibiting the peptidyl-prolyl cis±trans isomerase (PPIase) activity of FKBP12 and in promoting nerve regeneration both in vitro and in vivo [11].Given the therapeutic relevance of neurotrophic nonpeptidic molecules, we investigated the interaction of 1 and FKB...

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An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse

Cited by 99 publications

References 27 publications

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

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An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse

Cited by 99 publications

References 27 publications

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate,

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

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Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,