17 research outputs found
A microscale protein NMR sample screening pipeline
As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30â200Â ÎŒg in 8â35Â ÎŒl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure
Solid state carbon-13 NMR spectroscopy: the isomer distribution in the solution and solid phases of PdCl2(PMe2Ph)2
A solid-state 13C NMR spectrum, obtained with cross-polarization and magic-angle sample spinning, and a high-resoln. 13C NMR spectrum of a 0.07 M CHCl3 soln. indicated the presence of a 1:1 cis-trans ratio of the PdCl2(PMe2Ph)2 isomers in the solid-state. In EtOH soln. a 10:1 ratio was obsd. cis- And trans-PdCl2(PMePh2)2 also exhibited a 1:1 molar ratio in the solid state according to 13C NMR data. [on SciFinder (R)
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Sizable concentration-dependent frequency shifts in solution NMR using sensitive probes
Topoisomerase II-Drug Interaction Domains: Identification of Substituents on Etoposide that Interact with the Enzyme
Etoposide is one of the most successful chemotherapeutic agents used for the treatment of human cancers. The drug kills cells by inhibiting the ability of topoisomerase II to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. Etoposide is composed of a polycyclic ring system (rings AâD), a glycosidic moiety at the C4 position, and a pendant ring (Eâring) at the C1 position. Although drug-enzyme contacts, as opposed to drug-DNA interactions, mediate the entry of etoposide into the topoisomerase II-drug-DNA complex, the substituents on etoposide that interact with the enzyme have not been identified. Therefore, saturation transfer difference [1H]- nuclear magnetic resonance spectroscopy and protein-drug competition binding assays were employed to define the groups on etoposide that associate with yeast topoisomerase II and human topoisomerase IIα. Results indicate that the geminal protons of the Aâring, the H5 and H8 protons of the Bâring, as well as the H2â and H6â protons and the 3ââ and 5ââmethoxyl protons of the pendent Eâring interact with both enzymes in the binary protein-ligand complexes. In contrast, no significant nuclear Overhauser enhancement signals arising from the Câring, the Dâring, or the C4 glycosidic moiety were observed with either enzyme, suggesting that there is limited or no contact between these portions of etoposide and topoisomerase II in the binary complex. The functional importance of Eâring substituents was confirmed by topoisomerase II-mediated DNA cleavage assays
Tailoring <sup>1</sup>H Spin Dynamics in Small Molecules via Supercooled Water: A Promising Approach for Metabolite Identification and Validation
Metabolic mixtures are often analyzed via NMR spectroscopy
as it
provides a metabolic profile without sample alteration in a noninvasive
manner. These mixtures however tend to be very complex and demonstrate
considerable spectral overlap resulting in assignments that are sometimes
ambiguous given the range of current NMR methods available. De novo
molecular identification in these mixtures is generally accomplished
using chemical shift information and <i>J</i>-coupling based
experiments to determine spin connectivity information, but these
techniques fall short when a molecule of interest contains nonrelaying
centers. A method is presented here that enhances intramolecular spatial
interactions via supercooled water and uses the resulting spatial
correlations to edit mixtures. This is accomplished by utilizing nuclear
Overhauser effect spectroscopy (NOESY) at subzero temperatures in
capillaries to enhance NOE and provide more complete spin systems.
This technique is applied to a standard mixture of three known molecules
in D<sub>2</sub>O with overlapping resonances and is further demonstrated
to assign molecules in a worm tissue extract. The current method proves
to be a powerful complement to existing methods such as total correlation
spectroscopy (TOCSY) to expand the range of molecules that can be
assigned in situ without physical separation of mixtures
Three-Dimensional 13C-Detected CH3-TOCSY Using Selectively Protonated Proteins: Facile Methyl Resonance Assignment and Protein Structure Determination
Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627â636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357â406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585â592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR
Mandelalides AâD, Cytotoxic Macrolides from a New <i>Lissoclinum</i> Species of South African Tunicate
Mandelalides AâD are variously glycosylated, unusual
polyketide
macrolides isolated from a new species of <i>Lissoclinum</i> ascidian collected from South Africa, Algoa Bay near Port Elizabeth
and the surrounding Nelson Mandela Metropole. Their planar structures
were elucidated on submilligram samples by comprehensive analysis
of 1D and 2D NMR data, supported by mass spectrometry. The assignment
of relative configuration was accomplished by consideration of homonuclear
and heteronuclear coupling constants in tandem with ROESY data. The
absolute configuration was assigned for mandelalide A after chiral
GC-MS analysis of the hydrolyzed monosaccharide (2-<i>O</i>-methyl-α-l-rhamnose) and consideration of ROESY correlations
between the monosaccharide and aglycone in the intact natural product.
The resultant absolute configuration of the mandelalide A macrolide
was extrapolated to propose the absolute configurations of mandelalides
BâD. Remarkably, mandelalide B contained the C-4âČ epimeric
2-<i>O</i>-methyl-6-dehydro-α-l-talose. Mandelalides
A and B showed potent cytotoxicity to human NCI-H460 lung cancer cells
(IC<sub>50</sub>, 12 and 44 nM, respectively) and mouse Neuro-2A neuroblastoma
cells (IC<sub>50</sub>, 29 and 84 nM, respectively)
Computationally Assisted Discovery and Assignment of a Highly Strained and PANC-1 Selective Alkaloid from Alaska\u27s Deep Ocean
We report here the orchestration of molecular ion networking and a set of computationally assisted structural elucidation approaches in the discovery of a new class of pyrroloiminoquinone alkaloids that possess selective bioactivity against pancreatic cancer cell lines. Aleutianamine represents the first in a new class of pyrroloiminoquinone alkaloids possessing a highly strained multibridged ring system, discovered from Latrunculia ( Latrunculia) austini Samaai, Kelly & Gibbons, 2006 (class Demospongiae, order Poecilosclerida, family Latrunculiidae) recovered during a NOAA deep-water exploration of the Aleutian Islands. The molecule was identified with the guidance of mass spectrometry, nuclear magnetic resonance, and molecular ion networking (MoIN) analysis. The structure of aleutianamine was determined using extensive spectroscopic analysis in conjunction with computationally assisted quantifiable structure elucidation tools. Aleutianamine exhibited potent and selective cytotoxicity toward solid tumor cell lines including pancreatic cancer (PANC-1) with an IC50 of 25 nM and colon cancer (HCT-116) with an IC50 of 1 muM, and represents a potent and selective candidate for advanced preclinical studies