Journal of Biomolecular NMR

We present heteronuclear two-dimensional methods for the analysis of the geometry of exchangeable protons on a protein-bound carbohydrate. By using a water-selective NOESY-HSQC, we observed cross-relaxation between carbohydrate hydroxyl protons and non-exchangeable ring protons in the complex of [13C6]-α-methyl-D-mannopyranoside with recombinant rat mannose binding protein. Using a simple kinetic model, we were able to explain the differences in the initial slopes of the resulting cross-relaxation buildup curves in terms of the geometry of the hydroxyl protons in the bound state. The hydroxyl rotamers consistent with our cross-relaxation data fit very well with predictions based on the crystal structure of MBP bound to a mannose-rich oligosaccharide. These methods should be applicable to other systems where both ligand exchange and water exchange are fast relative to the rate of cross-relaxation.

The influenza virus fusion peptide is the N-terminal ~20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone 13CO-15N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct 13C shifts.

Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X4-Cys-X4-Cys-X28-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two α-helicies.

We demonstrate measurement of non-equilibrium backbone amide hydrogen–deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O2) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni2+-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.

A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.

The relaxation rates of the multiple-quantum coherence for the amide hydrogen of Gly13 in ras p21·GDP were determined in the presence and absence of 17O labeling in the β-phosphate of GDP. No significant difference could be observed between labeled and unlabeled samples, in spite of the fact that the hydrogen bond from the amide group of Gly13 to the β-phosphate is shorter than is typical, based on its chemical shift. For macromolecules in which an oxygen atom is the acceptor of a hydrogen bond, dipolar coupling between 17O and hydrogen is unlikely to be observable, except for extremely short H-bonds.