NMR Structure of Lipoprotein YxeF from Bacillus subtilis Reveals a Calycin Fold and Distant Homology with the Lipocalin Blc from Escherichia coli

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named ‘slim lipocalins’, with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses

Spatially Selective Heteronuclear Multiple‐Quantum Coherence Spectroscopy for Biomolecular NMR Studies

Spatially selective heteronuclear multiple‐quantum coherence (SS HMQC) NMR spectroscopy is developed for solution studies of proteins. Due to “time‐staggered” acquisitioning of free induction decays (FIDs) in different slices, SS HMQC allows one to use long delays for longitudinal nuclear spin relaxation at high repetition rates. To also achieve high intrinsic sensitivity, SS HMQC is implemented by combining a single spatially selective 1 H excitation pulse with nonselective 1 H 180° pulses. High‐quality spectra were obtained within 66 s for a 7.6 kDa uniformly 13 C, 15 N‐labeled protein, and within 45 and 90 s for, respectively, two proteins with molecular weights of 7.5 and 43 kDa, which were uniformly 2 H, 13 C, 15 N‐labeled, except for having protonated methyl groups of isoleucine, leucine and valine residues. Expect longer delays: Spatially selective (SS) HMQC NMR spectroscopy is presented for solution studies of proteins. Using SS HMQC allows one to employ long delays for longitudinal nuclear spin relaxation at high repetition rates for acquisition of free induction decays. This technique is applied to uniformly 13 C, 15 N‐labeled and uniformly 2 H, 13 C, 15 N‐labeled (but methyl group protonated) proteins with molecular weights of 7.5 and 43 kDa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107508/1/cphc_201301232_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/107508/2/1872_ftp.pd

GFT projection NMR for efficient H-1/C-13 sugar spin system identification in nucleic acids

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify H-1/C-13 sugar spin systems in C-13 labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of C-13-H-1 groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein

Highly Precise Measurement of Kinetic Isotope Effects Using ¹H‑Detected 2D [¹³C,¹H]-HSQC NMR Spectroscopy

A new method is presented for measuring kinetic isotope effects (KIEs) by ¹H-detected 2D [¹³C,¹H]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The high accuracy of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate dehydrogenase by comparing the 1-¹³C KIE with the published value obtained using isotope ratio mass spectrometry. High precision was demonstrated for the reaction catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-¹³C KIEs were found to be 1.0031(4), 1.0303(12), and 1.0148(2), respectively. These KIEs provide evidence for a cleanly rate-limiting retroaldol step during isomerization. The high intrinsic sensitivity and signal dispersion of 2D [¹³C,¹H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D ¹³C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for ¹H detection with cryogenic probes

Organotellurium Fluorescence Probes for Redox Reactions: 9‑Aryl-3,6-diamino­telluro­xanthylium Dyes and Their Telluroxides

Several 9-aryl-3,6-diamino­telluro­xanthylium dyes with phenyl, 2-methylphenyl, and 2,4,6-trimethylphenyl substituents at the 9-position were prepared. The characterization of these compounds included determination of ¹²⁵Te NMR spectra, fluorescence quantum yields (Φ_F), and quantum yields for the generation of singlet oxygen [Φ­(¹O₂)]. While these compounds were essentially nonfluorescent (Φ_F < 0.005), they produce ¹O₂ with Φ­(¹O₂) between 0.43 and 0.90. The tellurorosamines were oxidized with ¹O₂ via self-photosensitization to the corresponding telluroxides, which allowed their preparation free of excess oxidant. Telluroxides with a 9-(2-methylphenyl) or 9-(2,4,6-trimethylphenyl) substituent were fluorescent with quantum yields for fluorescence between 0.20 and 0.31. Steric bulk at the 9-position of the resulting telluroxides impacted rates of inter- and intramolecular attack of nucleophiles and stability of the telluroxide in aqueous media near physiological pH. The yield of reduction of the telluroxide with glutathione was also dependent on the steric bulk of the 9-aryl substituent. The structure of products from oxidation of the 9-(4-bromophenyl) tellurorosamine was determined by X-ray crystallography and indicated the addition of oxygen nucleophiles to the 9-position of the telluroxide oxidation state of the tellurorosamine

Comparison of <i>B. subtilis</i> YxeF NMR structure and <i>B. amyloliquefaciens</i> A7ZAF5 homology model.

<p>Surface electrostatic potential calculated for (A) the YxeF NMR structure (first conformer of ensemble deposited in the PDB) and (B) the homology model of A7ZAF5 by using the program GRASP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Petrey2" target="_blank">[56]</a> accessed through the protein function annotation server MarkUs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Petrey1" target="_blank">[55]</a>. The homology model was calculated using the SWISS-MODEL server in alignment mode <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Altschul1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Arnold1" target="_blank">[61]</a> and Verify3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Luthy1" target="_blank">[63]</a>, Procheck <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Laskowski1" target="_blank">[64]</a> and ProsaII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Sippl1" target="_blank">[65]</a> all atom z-scores (-1.12, −3.43 and −1.61, respectively) were obtained using the PSVS server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Bhattacharya2" target="_blank">[66]</a> and are indicative of a good quality model. In (C) and (D), ribbon drawings are shown for the structures of YxeF and A7ZAF5 in the same orientation, that is, viewed on the open end of the β-barrels. The acidic residues giving rise to the negative potential inside the cavities are depicted in licorice representation and are labeled (black for YxeF, red for A7ZAF5). (E) Pfam multiple alignment of the sequences of all members of PF11631. Except for YxeF (P54945), the sequences are labeled with their UniProt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-UniProt1" target="_blank">[25]</a> IDs (D4G3V0, E8VFY0, E0TYE6, D5MWC1, E3E109, A7ZAF5, E1UTS8). Amino acid background colors reflect average similarity inferred from the Blosum62 matrix, ranging from ‘most conserved’ (black) to ‘least conserved’ (white). YxeF and A7ZAF5 are highlighted in bold on the left and the region of the alignment used for building the comparative model of A7ZAF5 from the YxeF structure is enclosed by red boxes. The acidic residues labeled in (C) and (D) are marked with black (YxeF) and red (A7ZAF5) asterisks, respectively, above or below the alignment.</p

Comparison of β-barrels.

<p>Ribbon drawings of β-barrels of avidin (PDB ID 1AVD, green) in (A) and, after rotation by 180°, in (D); bacterial lipocalin Blc from <i>E. coli</i> (PDB ID 3MBT, orange) in (B) and (E); YxeF in (C) and (F) (PDB ID 2JOZ, blue). For clarity, the disordered terminal polypeptide segments of YxeF, as well as the corresponding segments in avidin and Blc, are not shown. In (A)–(C), β-strands A and H are labeled, while in (D)–(F) β-strand D is indicated.</p

Schematic representation of secondary structure element topologies.

<p>(A) YxeF, (B) lipocalins and (C) fatty acid-binding proteins. β-strands are represented by arrows, α-helices by rectangles, and 3₁₀-helices by ellipses. N- and C-termini are indicated as N and C respectively, and the ‘Ω-type’ loop L1 shared by YxeF and lipocalins is labeled.</p

Comparison of YxeF NMR structure (PDB ID 2JOZ, coded in blue) and Blc X-ray crystal structure (PDB ID 3MBT, orange).

<p>(A) Structure-based sequence alignment between YxeF and Blc obtained with the program DALI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Holm1" target="_blank">[16]</a>. The three structurally conserved regions (SCR1-3) typically found in lipocalins (see text) are boxed (continuous line for SCR1, which appears to be conserved in YxeF; dashed line for SCR2 and SCR3). Conserved residues being part of the calycin signature motif resulting in an interaction between Gly 36-X-Trp 38 in SCR1 and Arg 128 in SCR3 (see text) are highlighted using red boxes. Residues being part of the second hydrophobic core of Blc [see also (D] are highlighted using cyan boxes. (B) Superposition of the Trp and Arg residues being part of the calycin Gly-X-Trp and Arg motif in Blc (licorice representation, orange) and YxeF (line representation, all NMR conformers, blue). The superposition is obtained after superposition of the X-ray structure of Blc with each conformer of the NMR solution structure of YxeF (residues 32–132). (C) Structural superposition generated by the program DALI viewed from the open end of the β-barrels (for YxeF residues 32–132 were considered). In Blc, box 1 identifies the C-terminally located α-helix and box 2 the C-terminal β-strand, which are packed against the outside of the β-barrel and thereby form a second hydrophobic core (see D). (D) Ribbon drawing of the Blc structure with licorice representation of hydrophobic residues (in cyan) located in the C-terminal α-helix and on the outside of the β-barrel forming a second hydrophobic core [see also (C)].</p