Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

1H, 13C, and 15N chemical shift assignments of ZCCHC9

ZCCHC9 is a human nuclear protein with sequence homology to yeast Air1p/Air2p proteins which are RNA-binding subunits of the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex involved in nuclear RNA quality control and degradation in yeast. The ZCCHC9 protein contains four retroviral-type zinc knuckle motifs. Here, we report the NMR spectral assignment of the zinc knuckle region of ZCCHC9. These data will allow performing NMR structural and RNA-binding studies of ZCCHC9 with the aim to investigate its role in the RNA quality control in human.     

1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 (CaBP1)

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and a variety of voltage-gated Ca²⁺ channels in the brain. We report complete NMR chemical shift assignments of Ca²⁺-free CaBP1 (residues 1–167, BMRB no. 15197).     

1H, 15N, and 13C NMR chemical shift assignments for the Ig3 domain of palladin

As part of our NMR structure determination of the palladin Ig3 domain, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C, and ¹⁵N nuclei.     

1H, 15N, and 13C chemical shift assignments of calcium-bound calcium-binding protein 1 (CaBP1)

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and a variety of voltage-gated Ca²⁺ channels in the brain. We report complete NMR chemical shift assignments of Ca²⁺-bound CaBP1 (residues 1–167, BMRB no. 15623).     

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 chemical shift assignments of Ninjurin1 Extracellular N-terminal Domain

Cell adhesion molecules play a crucial role in fundamental biological processes via regulating cell–cell interactions. Nerve injury induced protein1 (Ninjurin1) is a novel adhesion protein that has no significant homology with other known cell adhesion molecules. Here we present the assignment of an 81 aa construct for human Ninjurin1 Extracellular N-Terminal (ENT) domain, which comprises the critical adhesion domain.     

1H, 15N,and 13C chemical shift assignments of murine calcium-binding protein 4

Calcium-binding protein 4 (CaBP4) regulates voltage-gated Ca²⁺ channels in retinal rod cells and specific mutations within CaBP4 are associated with congenital stationary night blindness type 2. We report complete NMR chemical shift assignments of the Ca²⁺-saturated form of CaBP4 with Ca²⁺ bound at EF1, EF3 and EF4 (BMRB no. 18877).     

1H, 13C,and 15N chemical shift assignments of neuronal calcium sensor protein,hippocalcin

Hippocalcin, a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, serves as an important calcium sensor for the slow afterhyperpolarizing (sAHP) current in the hippocampus, which underlies some forms of learning and memory. Hippocalcin is also a calcium sensor for hippocampal long-term depression (LTD) and genetically linked to neurodegenerative diseases. We report NMR chemical shift assignments of Ca²⁺-free hippocalcin (BMRB no. 18627).     

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).     

1H, 13C and 15N chemical shift assignments for an intracellular proteinase inhibitor of Bacillus subtilis

Intracellular proteinases (ISPs) are the main component of the bacilli degradome and a distinctive class in different bacilli. An intracellular proteinase inhibitor of the bacteria Bacillus subtillis was shown to regulate the activity of ISP-1. To study the structure of this inhibitor, we report the resonance assignment for this protein with 119 amino acid. The data will allow us to perform structural study on this inhibitor to understand its mechanism for ISP-1 inhibition.     

1H, 15N and 13C chemical shift assignments of the SH2 domain of human tensin2 (TENC1)

Tensin is an important cytoplasmic phosphoprotein localized to integrin-mediated focal adhesion. It links actin cytoskeleton to extracellular matrix through its N-terminal actin-binding domain and C-terminal phosphotyrosine-binding domain. Studies of knockout mice revealed the critical roles of tensin in skeletal muscle regeneration, renal function and regulation of cell migration. The SH2 domain of tensin interacts with various tyrosine-phosphorylated proteins thus functions as a platform for dis/assembly of signaling molecules. It has also been implicated in recruiting a tumor supperssor protein DLC1 (deleted in live cancer 1) to the focal adhesion, which is required for oncogenic inhibition effect of DLC1 in a phosphotyrosine-independent manner. Here, we report complete chemical shift assignments of the SH2 domain of human tensin2 determined by triple resonance experiments. The resonance assignments serve as a basis for our further functional studies and structure determination by NMR spectroscopy. (BMRB deposits with accession number 16472).     

1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast

The neuronal calcium sensor (NCS) proteins regulate signal transduction processes and are highly conserved from yeast to humans. We report complete NMR chemical shift assignments of the NCS homolog from fission yeast (Schizosaccharomyces pombe), referred to in this study as Ncs1p. (BMRB no. 16446).     

1H, 13C, and 15N chemical shift assignments for the RNA recognition motif of Nab3

Nuclear polyadenylated RNA-binding (Nab)3 protein is an RNA-binding protein that is involved in the poly(A) independent termination pathway. Here, we report the NMR spectral assignments of RNA-recognition motif (RRM) of Nab3. The assignment will allow performing NMR structural and RNA-binding studies of Nab3 with the aim to investigate its role in the poly(A) independent termination pathway.     

1H, 13C, and 15N resonance assignments for a ferrocytochrome c553 heme by multinuclear NMR spectroscopy

A novel strategy has been used to assign the 1H, 13C, and 15N resonances of the heme in Anabaena 7120 ferrocytochrome c553. 13C[13C] double-quantum coherence spectroscopy was used to delineate the heme carbons, 1H[13C] single-bond correlation spectroscopy was used to define the attached protons, and 1H[15N] multiple-bond correlation spectroscopy was used to assign the nitrogens. 1H[13C] multiple-bond correlation spectroscopy confirmed many of the assignments. Proteins were labeled uniformly with 13C or 15N to obtain the required spectral sensitivity.     

1H, 13C, and 15N backbone chemical shift assignments of StAR-related lipid transfer domain protein 5 (STARD5)

Steroidogenic acute regulatory (StAR)—related lipid transfer proteins possess a START (steroidogenic acute regulatory-related lipid transfer) domain. START domains are conserved protein modules involved in the non-vesicular intracellular transport of lipids and cholesterol in mammals. Fifteen mammalian proteins, divided in five subfamilies, are reported to possess a START domain. Members of the STARD4 subfamily, i.e. STARD4, 5 and 6 are essentially single START domains and are thought to be involved in the intracellular transport of cholesterol. No structure of a cholesterol-bound START domain from this family has been resolved yet. The determination of the structure of such a complex would contribute to a better understanding of the mechanism of ligand binding and transport by START domains, two unresolved aspects of their structural biology. In this context, we have undertaken the structure determination of a ligand-bound form of STARD5 by NMR. Here, we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments of the ligand-free STARD5.     

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).