NMR Facility Research Articles

Structure of the Mycobacterium Tuberculosis Virulence Factor Rv0899 (ompATb)

Rv0899 (OmpATb) is a 326-residue membrane-associated virulence factor of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. It is essential for the adaptation of Mtb to acidic environments and has been identified as an outer membrane protein. The M domain (residues 1-72) contains a 20-residue hydrophobic sequence that may form a membrane-anchoring helix. The B domain (residues 73-195) has been described as pore-forming. It shares sequence homology with the “BON” domain of Bacterial Osmotic-shock-resistance, Nodulation-specificity and lipid-binding proteins. The C domain (residue 196-326) shares significance homology with other bacterial peptidoglycan-binding domains, including the C-terminus of the E. coli outer membrane protein OmpA, after which Rv0899 was originally named. Using NMR spectroscopy, we show that residues 73-326, spanning the joint B and C domains, adopt a well-defined three-dimensional structure in water solution, however, the individual B and C domains fold independently and interact with each other to a minimal extent. The B domain adopts a rigid and stable structure, that forms a six-stranded β-sheet protected on one side by three α-helices. No comparable arrangement of secondary structure elements could be found in the databases, suggesting that is a new fold. The structure of the B domain is suggestive of a lipid binding or structural function, and provides insights to the role of Rv0899 in Mtb virulence.(This research was supported by the National Institutes of Health (NIH), and utilized the Burnham Institute NMR Facility supported by the NIH National Cancer Institute).

Tert-Butyl 3-(4-Cyano-5-phenylisothiazol-3-yl)carbazate

3-Iodo-5-phenylisothiazole-4-carbonitrile 1 reacts with tert-butyl carbazate via a Buchwald C-N style coupling to afford tert-butyl 3-(4-cyano-5-phenylisothiazol-3-yl)- carbazate 2 in 70% yield. [...]

Metals and membranes in neuroscience

The redox active transition metals Cu and Fe have been proposed as important factors in the neuropathology of Alzheimer’s disease (AD) and other neurodegenerative diseases. Although there is much experimental evidence on various aspects of the interaction between these metals and the molecular and supramolecular components of the neuropil, including the membranes, the structural biology of metal binding is very much a work in progress. The Australian Society for Biophysics (ASB) organized a one-day symposium on “Metals and membranes in neuroscience” as a satellite to the Seventh International Brain Research Organization (IBRO) World Congress of Neuroscience, held in Melbourne, Australia, in July 2007. Such a meeting in Australia was considered opportune with major new facilities coming on line, including expansion of the advanced EPR instrumentation at the Centre for Magnetic Resonance, University of Queensland; biological solidstate NMR at the Bio21 NMR Facility, University of Melbourne; neutron diVraction, scattering and reXectometry at the Bragg Institute associated with the new research reactor at the Australian Nuclear Science and Technology Organisation, Sydney; and XAFS, X-ray diVraction and X-ray scattering at the new Australian Synchrotron in Melbourne. Although the topic was treated from a biophysical viewpoint, the speakers had diverse backgrounds, ranging from neurobiology through quantum chemistry to molecular biology. The speakers, who are amongst the leading pioneers of metallo-neurobiology, have stimulated the interest of many biophysicists and structural biologists in the area and have contributed to this special issue of the European Biophysics Journal.

Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

Expanding your laboratory by accessing collaboratory resources.

The Environmental Molecular Sciences Laboratory (EMSL) in Richland, Washington, is the home of a research facility setup by the United States Department of Energy (DOE). The facility is atypical because it houses over 100 cutting-edge research systems for the use of researchers all over the United States and the world. Access to the lab is requested through a peer-review proposal process and the scientists who use the facility are generally referred to as ‘users’. There are six main research facilities housed in EMSL, all of which host visiting researchers. Several of these facilities also participate in the EMSL Collaboratory, a remote access capability supported by EMSL operations funds. Of these, the High-Field Magnetic Resonance Facility (HFMRF) and Molecular Science Computing Facility (MSCF) have a significant number of their users performing remote work. The HFMRF in EMSL currently houses 12 NMR spectrometers that range in magnet field strength from 7.05T to 21.1T. Staff associated with the NMR facility offers scientific expertise in the areas of structural biology, solid-state materials/catalyst characterization, and magnetic resonance imaging (MRI) techniques. The way in which the HFMRF operates, with a high level of dedication to remote operation across the full suite of High-Field NMR spectrometers, hasmore » earned it the name “Virtual NMR Facility”. This review will focus on the operational aspects of remote research done in the High-Field Magnetic Resonance Facility and the computer tools that make remote experiments possible.« less

Development of 1 GHz class NMR magnets

A development plan for 1 GHz class NMR magnets established and implemented at the Tsukuba Magnet Laboratory since 1995 is described. In addition, a NMR facility based on the Japanese genome science plan is introduced in relation to the 1 GHz NMR magnet project.

Nickel-Catalyzed Homoallylation of Aldehydes in the Presence of Water and Alcohols We thank Mr. Y. Ohhama, NMR Facility, for his outstanding technical assistance. Financial support from the Ministry of Education, Science, Sports, and Culture, Japanese Government, Grant-in-Aid for Scientific Research B, is acknowledged.

Nickel-Catalyzed Homoallylation of Aldehydes in the Presence of Water and Alcohols We thank Mr. Y. Ohhama, NMR Facility, for his outstanding technical assistance. Financial support from the Ministry of Education, Science, Sports, and Culture, Japanese Government, Grant-in-Aid for Scientific Research B, is acknowledged.

A G-Quartet Formed in the Absence of a Templating Metal Cation: A New 8-(N,N-dimethylaniline)guanosine Derivative This work was supported by the Robert A. Welch foundation. We thank Dr. Ben Shoulders and the staff at the NMR facility at the University of Texas for their assistance.

Contrary to what was assumed previously, self-assembled G-quartets may be obtained even in the absence of a templating alkali metal cation. This observation leads to the suggestion that this structural motif, which has been implicated in a variety of biological functions, might be more widespread than believed. Specifically, it is found that guanosine derivative 1 exists as a tetrameric ensemble both in chlorinated organic solvents and in the solid state (see picture). Ar=p-Me2N-C6H4, Rib=ribose. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2000/z13962_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Development and Use of a Virtual NMR Facility

We have developed a “virtual NMR facility” (VNMRF) to enhance access to the NMR spectrometers in Pacific Northwest National Laboratory's Environmental Molecular Sciences Laboratory (EMSL). We use the term virtual facility to describe a real NMR facility made accessible via the Internet. The VNMRF combines secure remote operation of the EMSL's NMR spectrometers over the Internet with real-time videoconferencing, remotely controlled laboratory cameras, real-time computer display sharing, a Web-based electronic laboratory notebook, and other capabilities. Remote VNMRF users can see and converse with EMSL researchers, directly and securely control the EMSL spectrometers, and collaboratively analyze results. A customized Electronic Laboratory Notebook allows interactive Web-based access to group notes, experimental parameters, proposed molecular structures, and other aspects of a research project. This paper describes our experience developing a VNMRF and details the specific capabilities available through the EMSL VNMRF. We show how the VNMRF has evolved during a test project and present an evaluation of its impact in the EMSL and its potential as a model for other scientific facilities. All Collaboratory software used in the VNMRF is freely available from www.emsl.pnl.gov:2080/docs/collab.

Structure of the Trinucleotide D-acp3U-A with Coordinated Mg2+Demonstrates that Modified Nucleosides Contribute to Regional Conformations of RNA

Abstract In tRNA crystal structures, the only nonaromatic ribonucleoside, dihydrouridine (D) and 3′-adjacent nucleotides adopt the infrequent 2′-endo conformation. Analysis of D, DUA and UUA by circular dichroism (CD) and NMR confirmed that D produces the 2′-endo conformation of the trimer and is responsible for the same in tRNA. The nucleoside, 3-[3-(S)-amino-3-carboxypropyl]uridine (acp3U) occurs in the tRNA sequence D-acp3U-A. CD spectra indicated that D-acp3U-A and U-acp3U-A bind Mg2+, whereas acp3U, D-acp3U, DUA and UUA do not. Ion dependent changes in chemical shifts and paramagnetic broadening of 1H signals indicated that Mg2+ coordination involved the acp3U side chain and the ribose of A. The Mg2+-bound structure was modeled with simulated annealing, molecular mechanics and NMR restraints. Acp3U contributed to local charge density that facilitated Mg2+ coordination. #Present addresses: W. W. S., NMR Facility University of California, Davis, CA; B. F., School of Medicine, University of North Caroli...

DIASTEREOISOMERS FROM IODINE-INDUCED CYCLIZATION REACTION OF PHOSPHONATE

Abstract Two products were isolated from the iodine-induced cyclization reaction of di-n-propyl-4-pentenyl-phosphonate, 1. One of them has previously been proven to be 2-n-propyloxy-2-oxo-6-(iodomethyl)-1,2-oxaphosphorinane, 2. The second product, 3, has the same molecular weight and mass spectral fragmentation patterns as 2. The results of one- and two-dimensional 1H. 13C. and 31P multinuclear magnetic resonance spectroscopies indicate that 2 and 3 are the alkoxyl-axial and alkoxyl-equatorial diastereoisomers, respectively, of 2-n-propyloxy-2-oxo-6-(iodomethyl)-1, 2-oxaphosphorinane. The nuclear magnetic resonance spectroscopic parameters such as the 1H, l3C, and 31P chemical shifts and the homo-(1H–1H) and hetero-(1H–13C, lH 31P, and 13C – 31P)coupling constants of 2, and 3 are documented in this report. All NMR spectra were obtained from the Biophysics NMR Facility Center, Johns Hopkins University, established by a N.I.H. grant GM-27512 and the work was also partially supported by a N.I.H. grant GM 34252 (to LSK). We wish to thank Dr. Daniel Callahan and Mr. Chang Lee for editorial assistance, and the grant from the Chinese National Science Foundation.

Rearrangements of cyclobutenones. Conversion of selected 4-allylcyclobutenones to bicyclo[3.2.0]heptenones

J . Org. Chem. 1989,54, 6018-6021 Scheme I11 TEST H --Co2Et v OAc c K-PH1 5 HI 1 x=o .c-- fi OAc X =aOAc;PH (i) DiBAH c (ii) Ph3P = CH (CH& QK from 12. The overall yield of 1 from 3 is 27%. As pre- viously described, 35J2 is readily available in enantiomer- ically homogeneous form from cis-l,4-diacetoxycyclo- pentene. The stereochemical outcome a t C13 arising from the coupling of similar substrates under the same conditions is amazing. In the previously described aldols5 leading to 5 and 6 no other stereoisomers were observed. Yet when the ynal 7 is employed 8 is the only product observed! Instead of offering ad hoc interpretations that do not flow from sound experimental observations, we prefer to outline an agenda of questions that must be addressed. Why does a particular substitution type on the aldehyde favor or disfavor silyl transfer? Is silyl transfer fundamental or accessory to the stereochemical outcome? Do the sharply differing results arise from a common transition-state (12) Cf: (a) Deardorff, D. R.; Myles, D. C.; MacFerrin, K . D. Tetra- hedron Lett. 1985, 5615. (b) Deardorff, D. R.; Matthews, A. J.; McMeekin, D. S.; Craney, C. L. Ibid. 1986, 1255. alignment differing in the placement of the R and H group of the aldehyde or do the reaction types differ in overall topography (chair vs boat, synclinal vs antiperiplanar)? Answers to these sorts of questions are not readily obtained but are crucial to illuminating this interesting stereo- chemical finding and extending it to new domains. I n the meantime, we note that the chemistry disclosed here and previously5 provides straightforward access to optically pure prostaglandins with complete control of the con- figuration at either C13 or C15 in any stereochemical sense. Acknowledgment. This research was supported by PHS Grant HL25848. NMR Spectra were obtained through the auspices of the Northeast Regional NSF/ NMR facility at Yale University, which was supported by NSF Chemistry Division Grant CHE 7916210. Supplementary Material Available: Experimental proce- dures and characterization data for all compounds (5 pages). Ordering information is given on any current masthead page. Rearrangements of Cyclobutenones. Conversion of Selected 4-Allylcyclobutenones to Bicycle[ 3.2.0lheptenones Simon L. Xu and Harold W . Moore* Department of Chemistry, University of California, Iruine, California 9271 7 Received October 9. 1989 Summary: 4-Allyl-4-alkoxy(or hydroxy or (trimethyl- sily1)oxy)cyclobutenones are reported to rearrange to bi- cyclo[3.2.0] hept-2-en-7-ones upon thermolysis in refluxing toluene. The synthetic scope and mechanism of this un- usual transformation are discussed. The products are envisaged to arise from an electrocyclic ring opening of the cyclobutenones to the corresponding vinylketenes which then undergo an intramolecular [ 2 21 cycloaddition of the ketene moiety to the nonconjugated allylic double bond. Sir: Selected 4-alkynyl, 4-alkenyl-, and 4-arylcyclo- butenones have recently been shown to undergo facile ring expansion to respectively benzoquinones, hydroquinones, 0 1989 American Chemical Society

Covalent binding of a fluorinated 2-nitroimidazole to emt-6 tumors in Balb/C mice: detection by f-19 nuclear magnetic resonance at 2.35 T

A hexafluorinated 2-nitroimidazole (CCI-103F) has been synthesized and its properties as a hypoxic cell binder investigated. The drug has a plasma half-life of 90 min in Balb/C mice and a tumor-to-plasma ratio of 0.8. Following sustained exposure to the drug and a washout period for the unbound drug, liver and tumor samples were excised NMR investigation of the excised tissue in a 2.35 T animal sized NMR facility revealed the presence of 19F bound to both tissues. Further improvement in sensitivity is required to make 19F NMR detection of binding in situ feasible.

New NMR spectrometer doubles resolving power

A new, high‐resolution, 600‐MHz nuclear‐magnetic‐resonance spectrometer at Carnegie‐Mellon University will be ready for experimenters early in the spring. With a magnetic field almost twice that of previously available NMR spectrometers, the new spectrometer provides roughly a doubling of resolving power for the study of organic molecules. The Carnegie‐Mellon NMR Facility for Biomedical Studies (a joint undertaking of Carnegie‐Mellon and the University of Pittsburgh) has called for proposals for a first round of short experiments covering a broad spectrum of applications, to explore the capabilities of the spectrometer.