Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.     

Assignment of 1H, 13C and 15N resonances of N-terminal domain of DnaA protein

DnaA protein is a key protein in the initiation of chromosomal replication in Escherichia coli. We reported the assignments of ¹H, ¹³C, and ¹⁵N resonances of N-terminal domain of Dna A (1–108) which contains the activities of self-oligomerization and DnaB helicase loading.     

13C and proton NMR studies of horse cytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of the 55 methyl groups of horse cytochrome c have been determined over a range of temperatures both in the diamagnetic ferrocytochrome and in the paramagnetic ferricytochrome. Specific assignments of many proton resonances have been published previously and all of the remaining methyl proton resonances are now specifically assigned. The corresponding 13C assignments follow directly, including those of contact shifted 13C resonances which are reported for the first time.     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein

This note presents the ¹H, ¹³C, and ¹⁵N resonances assignment of the 22 kDa, dimeric, C-repressor protein from the P2 bacteriophage. The C-repressor controls the genetic switch that determines if the temperate P2 phage should exist in the lytic or lysogenic lifemode.     

Crystallization of tuna ferricytochrome c at low ionic strength

Previous crystallographic studies of tuna ferricytochrome c have employed crystals grown from solutions of ammonium sulfate, corresponding to an ionic strength of 9.5 M (Takano, T., and Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115). To obtain a structure at a lower ionic strength, the ferric tuna protein was crystallized at neutral pH with polyethylene glycol at an ionic strength of 45 mM, These crystals (space group P2(1), a = 37.11 A, b = 107.66 A, c = 55.75 A, beta = 105.3 degrees) contain four molecules/asymmetric unit and grow to dimensions of 0.2 X 0.4 X 1.0 mm in 2-4 weeks. They diffract to beyond 1.8 A and are stable in the x-ray beam. We have recorded 28,198 unique Bragg reflections (83% of those possible) to a resolution of 1.89 A from a native crystal. We are undertaking a solution of this structure by the molecular replacement method.     

13C-NMR studies of horse ferrocytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of 53 of the 55 methyl resonances of horse ferrocytochrome c have been determined by editing natural abundance 13C spectra according to the number of attached protons, observing the temperature dependence of the chemical shifts, and correlating 13C and proton chemical shifts in two-dimensional spectra. Previous assignments of proton shifts allow 16 of the 13C resonances to be assigned firmly.     

A new assignment strategy for the hyperfine-shifted 13C and 15N resonances in Fe2S2 ferredoxins

Assignment of hyperfine-shifted resonances in paramagnetic metalloproteins such as Fe2S2 ferredoxins poses a major experimental challenge due to hyperfine shifts and/or severe line broadening. We have explored the possibility of using structural data from homologous proteins as part of an assignment strategy for the sequence-specific assignment of hyperfine-shifted backbone carbonyl (13C') and nitrogen resonances (15N) in Fe2S2 ferredoxins. This strategy is based on the assignment of resonances in the paramagnetic region to particular types of amino acid residues using selective isotope labeling. Reduced metal-nuclear distances are then calculated from experimentally determined T1 relaxation times for those resonances and the calculated distances aligned with the distances of nuclei at corresponding amino acid sequence positions in the crystal structure of a structurally homologous protein. The comparative assignment approach has met with success in correctly predicting the 13C' and 15N assignments in Pdx degrees from the crystal structure data of two similar and related ferredoxins, namely bovine adrenodoxin and Anabaena ferredoxin. Sequence-specific assignments made in this fashion were verified by selective 13C'{15N} decoupling experiments.     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240

The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The ¹H chemical shifts of complexed L-161,240 are also determined.     

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.