Backbone and sidechain 1H, 15N and 13C assignments of the NLRP7 pyrin domain

The resonance assignments of the human NLRP7 PYD domain have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the 3D structure determination of the NLRP7 PYD domain.     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

1H, 13C and 15N resonance assignments for the human E2 conjugating enzyme, UbcH7

UbcH7 is a human E2 conjugating enzyme in the ubiquitin-dependent protein degradation pathway. The resonance assignments of UbcH7 will assist in elucidating the structural basis of interactions that occur within ubiquitination.     

Evaluation of Live Attenuated H7N3 and H7N7 Vaccine Viruses for Their Receptor Binding Preferences,Immunogenicity in Ferrets and Cross Reactivity to the Novel H7N9 Virus

Live attenuated influenza vaccine (LAIV) candidates of the H7 subtype, A/Netherlands/219/03 (H7N7, NL03 ca) and A/chicken/British Columbia/CN-6/2004 (H7N3, BC04 ca), were evaluated for their receptor binding specificity and immunogenicity in ferrets. The BC04 ca virus exhibited α2,3-SA and α2,6-SA dual receptor binding preference while the NL03 ca virus preferentially bound to α2,3-SA. Substitution of the Q226 and G228 (Q-G) by the L226 and S228 (L-S) residues in the HA improved binding to α2,6-SA for NL03 ca. The vaccine viruses with L-S retained the attenuation phenotype. NL03 L-S ca replicated more efficiently than the original NL03 ca virus in the upper respiratory tract of ferrets, and induced higher levels of humoral and cellular immune responses. Prior vaccination with seasonal LAIV reduced H7-specific antibody responses, but did not reduce the H7N7 vaccine mediated protection against a heterologous H7N3 BC04 wt virus infection in ferrets. In addition, the H7N3 and H7N7 vaccine immunized ferret sera cross reacted with the newly emerged H7N9 virus. These data, in combination with the safety data from previously conducted Phase 1 studies, suggest that these vaccines may have a role in responding to the threat posed by the H7N9 virus.     

1H, 13C, and 15N assignment of the muscular LIM protein MLP/CRP3

The family of CRP proteins comprises three members, which are composed of two LIM domains separated by a long linker of more than 50 residues. We determined the structure of the muscle variant, MLP (CRP3), by nuclear magnetic resonance and show that the two LIM domains are independent of each other.     

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HA_m) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HA_m constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.     

Exposure to a Low Pathogenic A/H7N2 Virus in Chickens Protects against Highly Pathogenic A/H7N1 Virus but Not against Subsequent Infection with A/H5N1

Recent evidences have demonstrated that the presence of low pathogenic avian influenza viruses (LPAIV) may play an important role in host ecology and transmission of avian influenza viruses (AIV). While some authors have clearly demonstrated that LPAIV can mutate to render highly pathogenic avian influenza viruses (HPAIV), others have shown that their presence could provide the host with enough immunological memory to resist re-infections with HPAIV. In order to experimentally study the role of pre-existing host immunity, chickens previously infected with H7N2 LPAIV were subsequently challenged with H7N1 HPAIV. Pre-infection of chickens with H7N2 LAPIV conferred protection against the lethal challenge with H7N1 HPAIV, dramatically reducing the viral shedding, the clinical signs and the pathological outcome. Correlating with the protection afforded, sera from chickens primed with H7N2 LPAIV reacted with the H7-AIV subtype in hemagglutination inhibition assay and specifically with the N2-neuraminidase antigen. Conversely, subsequent exposure to H5N1 HPAIV resulted in a two days-delay on the onset of disease but all chickens died by 7 days post-challenge. Lack of protection correlated with the absence of H5-hemagglutining inhibitory antibodies prior to H5N1 HPAIV challenge. Our data suggest that in naturally occurring outbreaks of HPAIV, birds with pre-existing immunity to LPAIV could survive lethal infections with HA-homologous HPAIV but not subsequent re-infections with HA-heterologous HPAIV. These results could be useful to better understand the dynamics of AIV in chickens and might help in future vaccine formulations.     

Excited-state relaxation paths of oxo/hydroxy and N9H/N7H tautomers of guanine: a CC2 theoretical study

We performed a theoretical investigation, at the CC2/aug-cc-pVDZ level, of the ring-deformation mechanisms of four guanine tautomers (oxo, hydroxy, N9H, and N7H). The study showed that the optimized conical intersections S₀/S₁ are accessible through the ¹ππ* excited states of tautomers. The optimized conical intersections S₀/S₁, which show deformation at the pyrimidine ring, have high energies. This means that the relaxations of the ¹ππ* excited states via internal conversion are disfavored. For two tautomers we found crossing points ¹ππ*/¹πσ* of the excited-state reaction paths, revealing the possibility of a population of the ¹πσ* excited state by the ¹ππ* excited state.

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

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, 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 resonance assignments of the Calmodulin-Munc13-1 peptide complex

Ca²⁺-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca²⁺-Calmodulin/Munc13-1^458–492 peptide complex at pH 6.8 and 35°C (BMRB No. 15470).     

1H, 13C and 15N resonance assignments of the complement control protein modules of the complement component C7

Human C7 is one of four homologous complement proteins that self-assemble on the nascent activation-specific fragment, C5b, thus forming the cytolytic membrane attack complex (MAC). In addition to the conserved modular core of the MAC/perforin protein family, C7 has four C-terminal domains comprising a pair of complement control protein modules (CCPs) preceding two Factor-I like modules (FIMs). It is proposed that the C7-CCPs might serve as a molecular arm for delivery of C7-FIMs to their binding site on C5b. Here we present the NMR chemical shift assignments for the C7-CCPs produced as a 14-kDa recombinant protein. Based upon triple-resonance experiments, 98 and 94 % of the backbone and side-chain (¹H, ¹³C and ¹⁵N) assignments, respectively, have been completed. The chemical shifts and assignments have been deposited in the BioMagResBank database under accession number 18530.