1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase

The 17.7 kDa R2 module from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been isotopically labeled (¹³C,¹⁵N) and recombinantly expressed. Here we report the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R2.     

NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been ¹³C, ¹⁵N isotopically labelled and recombinantly expressed. We report here the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R3.     

Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

Alginate epimerases are large multidomain proteins capable of epimerising C5 on β‐D ‐mannuronic acid (M) turning it into α‐L ‐guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution‐state NMR are hampered by their size—the smallest epimerase, AlgE4, consisting of one A‐ and one R‐module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally ²H, ¹⁵N labeled AlgE4 isotopomeres (A‐[²H, ¹⁵N]‐R and [²H, ¹⁵N]‐A‐R) by protein trans‐splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild‐type AlgE4.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).     

1H, 15N and 13C resonance assignments,secondary structure,and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase

Summary By using fully ¹⁵N- and ¹⁵N/¹³C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific ¹H and ¹⁵N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the ¹³C resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected ¹⁵N and ¹³C experiments. A smaller but significant subset of side-chain ¹H and ¹³C assignments were also determined. In this complex, only one ¹⁵N or ¹³C resonance was detected per ¹⁵N or ¹³C protein nucleus, which indicated a single conformation. Proton-detected ¹³C experiments were also performed with unlabeled DHFR, complexed with ¹³C-7/¹³C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on ¹H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667–9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.     

Secondary structure and 1H, 13C and 15N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli

We report the ¹H, ¹³C and ¹⁵N backbone chemical shift assignments and secondary structure of the Escherichia coli protein BamC, a 32-kDa protein subunit that forms part of the BAM (Omp85) complex, the β-barrel assembly machinery present in all Gram-negative bacteria and which is essential for viability.     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

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.     

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The ¹H, ¹³C, and ¹⁵N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C.     

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, 13C, 15N resonance assignment of the chitin-binding protein CBP21 from Serratia marcescens

The 18.8 kDa chitin-binding protein CBP21 from Serratia marcescens has been isotopically labeled and recombinantly expressed. In this paper, we report the ¹H, ¹³C, ¹⁵N resonance assignment of CBP21.     

1H, 13C,and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin

The ¹H, ¹³C, and ¹⁵N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-¹⁵N] FKBP/ascomycin and [U-¹⁵N, ¹³C] FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, ³J coupling constants, amide exchange data, and the differences between the C^α and C^β chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution. © 1993 John Wiley & Sons, Inc.     

1H, 13C and 15N chemical shift referencing in biomolecular NMR

Summary A considerable degree of variability exists in the way that ¹H, ¹³C and ¹⁵N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common ¹H, ¹³C and ¹⁵N chemical shift standards, now used in biomolecular NMR, to those proposed here.Abbreviations TMS tetramethylsilane - TSP 3-(trimethylsilyl)-propionate, sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - TFE 2,2,2-trifluoroethanol - DMSO dimethyl sulfoxide     

1H, 13C and 15N resonance assignments and peptide binding site chemical shift perturbation mapping for the Escherichia coli redox enzyme chaperone DmsD

Herein are reported the mainchain ¹H, ¹³C and ¹⁵N chemical shift assignments and amide ¹⁵N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein.     

Sequence-specific 1H, 15N and 13C resonance assignments of Art v 1: a proline-rich allergen of Artemisia vulgaris pollen

Art v 1 is the major allergen of Artemisia vulgaris. The IgE raised against Art v 1 not only can cross-react with other proteins from the Asteraceae family members but also with components of various forms of food. Art v 1 is an important target for immunotherapy strategies, including vaccination with hypoallergenic derivatives or chimeras. We report the ¹H, ¹³C, and ¹⁵N resonance assignments of the recombinant Art v 1 and identification of secondary structures based on ¹³C chemical shifts.     

1H, 15N and 13C chemical shift assignments of the Cdt1 binding domain of human Mcm6

The eukaryotic minichromsome maintenance (Mcm) proteins (Mcm2–7) are evolutionally conserved from yeast to human. These proteins are essential for DNA replication and Mcm6 is one subunit of Mcm2–7 complex that serves as the replicative helicase in DNA replication. Cdt1 is a critical member of pre-replicative complex (pre-RC), which directs the chromatin loading of Mcm2–7 complex. The Cdt1 binding domain (CBD) of human Mcm6 was found to directly interact with Cdt1 and this interaction may mediate the chromatin loading of Mcm2–7 complex. The structure of CBD exhibits a typical “winged-helix” fold which is generally involved in protein-nucleic acid interaction. Here we report the ¹H, ¹⁵N and ¹³C chemical shift assignments of human Mcm6 CBD determined by triple resonance experiments. The resonance assignments obtained in this work were required for the structure–function studies of CBD by NMR spectroscopy (BMRB deposits with accession number 16396).     

Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H+-ATP synthase subunit c, by magic-angle spinning solid-state NMR

Signal assignment and secondary structural analysis of uniformly [¹³C, ¹⁵N] labeled H⁺-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the ¹³C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue ¹³C–¹³C and ¹³C–¹⁵N chemical shift correlations, 78% of , 72% of , 62% of C′ and 61% of N^H signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the ¹³C–¹³C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by -helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.     

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).     

Secondary structure and 1H, 13C and 15N resonance assignments of BamE, a component of the outer membrane protein assembly machinery in Escherichia coli

We report the ¹H, ¹³C and ¹⁵N backbone and side chain chemical shift assignments and secondary structure of the Escherichia coli protein BamE, a subunit of the BAM (Omp85) complex, the β-barrel assembly machinery present in all Gram-negative bacteria, mitochondria and chloroplasts and is essential for viability.