UVI31+ Is a DNA Endonuclease That Dynamically Localizes to Chloroplast Pyrenoids in C. reinhardtii

UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.     

1H, 13C, and 15N resonance assignment of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis

We report the nearly complete ¹H, ¹³C, and ¹⁵N resonance assignments of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitides. Secondary structure determination using CSI method leads to the prediction of nine β-sheet parts.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of an unusual Ca<Superscript>2+</Superscript>-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> in its apo form

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of an unusual Ca²⁺-binding protein from Entamoeba histolytica (EhCaBP6) in its apo form as a prelude to its structural and functional characterization.     

1H, 13C and 15N resonance assignments of wild-type Bacillus subtilis Lipase A and its mutant evolved towards thermostability

Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete ¹H, ¹³C and ¹⁵N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a calcium-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization.     

1H, 13C, and 15N resonance assignment of photoactive yellow protein

Photoactive yellow protein (PYP) is involved in the negative phototactic response towards blue light of the bacterium Halorhodospira halophila. Here, we report nearly complete backbone and side chain ¹H, ¹³C and ¹⁵N resonance assignments at pH 5.8 and 20 °C of PYP in its electronic ground state.     

1H, 13C, and 15N NMR assignments of StnII-Y111N, a highly impaired mutant of the sea anemone actinoporin Sticholysin II

Sticholysin II is an actinoporin of 175 amino acids produced by the sea anemone Stichodactyla helianthus. Several studies with different mutants have been performed to characterize its molecular properties and activity. As a first step towards a 3D structural characterization and its interaction with membrane models at a residue level, herein we report the nearly complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of the Y111N variant at pH 4.0 and 25°C (BMRB No. 16630). The assignment is complete for the biologically relevant residues, specially for those implicated in membrane interactions.     

Sequence specific <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a cataract-related variant G57W of human γS-crystallin

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [¹⁵N–¹H]-HSQC spectrum recorded with uniformly ¹³C/¹⁵N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.     

3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-²H,¹⁵N,¹³C; Ile??1-{¹³CH₃}; Leu,Val-{¹³CH₃/¹²CD₃}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-??YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40?°C to improve the sensitivity of the measurements and alleviate the overlap of ¹H?C¹⁵N correlations in the abundant ??-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of ¹H?C¹⁵N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D ¹H?C¹⁵N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected ??out-and-back?? HMCM(CG)CBCA experiment, and (3) strong sequential HN?CHN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578?C195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.     

1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) of Escherichia coli. Virtually complete (98%) backbone 1H, 15N, and 13C nuclear magnetic resonance (NMR) signal assignments were determined by using a battery of triple-resonance three-dimensional (3D) NMR pulse sequences. In addition, nearly complete (1H, 95%; 13C, 85%) side-chain 1H and 13C signal assignments were obtained from an analysis of 3D 13C HCCH-COSY and HCCH-TOCSY spectra. These experiments rely almost exclusively upon one- and two-bond J couplings to transfer magnetization and to correlate proton and heteronuclear NMR signals. Hence, essentially complete signal assignments of this 168-residue protein were made without any assumptions regarding secondary structure and without the aid of a crystal structure, which is not yet available. Moreover, only three samples, one uniformly 15N-enriched, one uniformly 15N/13C-enriched, and one containing a few types of amino acids labeled with 15N and/or 13C, were needed to make the assignments. The backbone assignments together with the 3D 15N NOESY-HMQC and 13C NOESY-HMQC data have provided extensive information about the secondary structure of this protein [Pelton, J.G., Torchia, D.A., Meadow, N.D., Wong, C.-Y., & Roseman, S (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3479-3488]. The nearly complete set of backbone and side-chain atom assignments reported herein provide a basis for studies of the three-dimensional structure and dynamics of IIIGlc as well as its interactions with a variety of membrane and cytoplasmic proteins.     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Backbone 1H, 13C and 15N resonance assignments of an intrinsically unstructured βγ-crystallin from Hahella chejuensis

The sequence specific backbone ¹H, ¹³C and ¹⁵N resonance assignments of an intrinsically unstructured βγ-crystallin from Hahella chejuensis are reported. The secondary structure chracterization of the unstructured protein reveals that large fraction of residues exhibits β-strand propensity, as in the case of the Ca²⁺-bound structured protein.     

1H, 13C and 15N backbone and side-chain chemical shift assignments for oxidized and reduced desulfothioredoxin

Based on sequence homology, desulfothioredoxin (DTrx) from Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thioredoxin superfamily. Desulfothioredoxin (104 amino acids) contains a particular active site consensus sequence, CPHC probably correlated to the anaerobic metabolism of these bacteria. We report the full ¹H, ¹³C and ¹⁵N resonance assignments of the reduced and the oxidized form of desulfothioredoxin (DTrx). 2D and 3D heteronuclear NMR experiments were performed using uniformly ¹⁵N-, ¹³C-labelled DTrx. More than 98% backbone and 96% side-chain ¹H, ¹³C and ¹⁵N resonance assignments were obtained. (BMRB deposits with accession number 16712 and 16713).     

Sequential assignments in uniformly 13C- and 15N-labelled RNAs: The HC(N,P) and HC(N,P)-CCH-TOCSY experiments

Summary An approach for the simultaneous acquisition of HCN and HCP as well as HCN-CCH-TOCSY and HCP-CCH-TOCSY triple resonance data sets for ¹³C-/¹⁵N-labelled RNAs is presented. The new HCN-CCH-TOCSY scheme unambiguously links all sugar resonances to the base nitrogen. In addition, simultaneous acquisition of HCN-CCH-TOCSY and HCP-CCH-TOCSY data sets provides sequential and base-type information in a single experiment, thereby saving data acquisition time as well as providing complementary data sets that are useful in clarifying ambiguous assignments. Virtually complete sequence-specific phosphate-ribose ¹H, ³¹P, and base ¹⁵N1,9 assignments as well as partial ¹³C assignments could be obtained in a single experiment for a 0.5-mM sample of a 19-mer ribonucleotide.     

1H, 13C, and 15N NMR assignments of StnII-R29Q, a defective lipid binding mutant of the sea anemone actinoporin Sticholysin II

Several studies have been made to characterize the molecular properties and activity of Sticholysin II (StnII), a 175 amino acid protein secreted by the sea anemone Stichodactyla helianthus. In particular, the biochemical characterization of different mutants of this protein have been shown to be essential for the rational understanding of its activity. Here we report the nearly complete NMR ¹⁵N, ¹³C and ¹H chemical shift assignments, at pH 4.0 and 25°C, of a less hemolytic and defective lipid binding mutant of StnII, the R29Q variant (BMRB no 16362).     

Aliphatic 1H, 13C and 15N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP+ and folate

Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been ¹³C/¹⁵N isotopically labelled and purified. Here, we report the aliphatic ¹H, ¹³C and ¹⁵N resonance assignments of MpDHFR in complex with NADP⁺ and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.     

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1α

As part of our NMR structure determination of the human Interleukin-1α, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C and ¹⁵N nuclei.