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.     

Backbone (15)N relaxation analysis of the N-terminal domain of the HTLV-I capsid protein and comparison with the capsid protein of HIV-1

Human T-cell leukemia virus type 1 (HTLV-I) is an oncogenic retrovirus that exhibits specific tropism for human T-cells. The capsid (CA) proteins of retroviruses share highly conserved secondary and tertiary structures. However, they can form quaternary structures (assembled cores) that are conical (e.g., the lentivirus subgroup, including HIV) or spherical (e.g., the oncovirus subgroup, including HTLV). The intrinsic features that drive these differences are not understood. So far, only structural studies have been used as a basis for comparison. Dynamics may play a role in particle formation. High-resolution nuclear magnetic resonance (NMR) (15)N relaxation data (T(1), T(1rho), and NOE) have been used to characterize the backbone dynamics of the N-terminal domain (NTD) of the oncovirus HTLV-I and to compare with the CA NTD of HIV-1. Large variations in the (15)N heteronuclear NOEs and transversal relaxation rates for individual residues are consistent with the bundle RMSD of the previously calculated NMR structures. The beta-hairpin and CyP-A loop exhibit different mobility in HTLV-I and HIV-1. The overall hydrodynamic property of the HTLV-I capsid NTD is quite distinct from the HIV-1.     

Sequence-specific 1H, 13C, and 15N resonance assignment of the autophagy-related protein Atg8

The autophagy-related protein Atg8 is important for the formation of autophagosomes as it mediates membrane fusion. To elucidate the solution structure of Atg8 backbone and side chain chemical shifts of Atg8 were assigned as far as possible.