All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp 21 -Tyr 22 -Gly 23 . The heteronuclear triple resonance NMR study reported here of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline, reveals a remarkably tight helical hairpin structure, with its N-terminal α-helix (Gly 1 -Gly 12 ) packed tightly against its second α-helix (Trp 14 -Gly 23 ), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive ϕ angle, enabling the hairpin turn that links the two helices. The structure is stabilized by multiple interhelical C α H to C¼O hydrogen bonds, characterized by strong interhelical H N -H α and H α -H α NOE contacts. Many of the previously identified mutations that make HA2 nonfusogenic are also incompatible with the tight antiparallel hairpin arrangement of the HAfp helices. 15 N relaxation analysis indicates the structure to be highly ordered on the nanosecond time scale, and NOE analysis indicates HAfp is located at the water-lipid interface, with its hydrophobic surface facing the lipid environment, and the Gly-rich side of the helix-helix interface exposed to solvent. Of the three different types of RNA influenza virus, the influenza A type is the most common and virulent human pathogen. Based on the antibody response to its viral surface proteins, hemagglutinin and neuraminidase, influenza type A is further subdivided into serotypes, ranging from subtypes H1-16 for hemagglutinin, and N1-9 for neuraminidase. Subtypes H1, H2, H3, N1, and N2 are the most common in human disease (1). Whereas neuraminidase is an enzyme responsible for cleavage of cellular sialic acid carbohydrates, a key step in viral exit, HA mediates cellular entry through binding to carbohydrates on epithelial cells and subsequent delivery of the viral contents to the cellular interior (2, 3).Prior to infection of human tissue, the homotrimeric hemagglutinin, HA0, requires cleavage by one of several human proteases (4), resulting in a trimer of heterodimeric proteins, HA1 and HA2, covalently linked to one another by a single disulfide bridge. The atomic structures of the precursor and processed HA have been solved by X-ray crystallography (5-7) and reveal that the main rearrangement taking place upon cleavage is the relocation of the newly formed N-terminal HA2 tail from the surface of HA0 to the helical core of the HA2 ecto domain. Upon lowering the pH, the N-terminal HA2 fusion domain is extruded from the HA2 helical core to a position where it can target the endosomal membrane, while the C-terminal transmembrane helix keeps HA2 anchored to the viral membrane. ...

The majority of protein structures are determined in the crystalline state, yet few methods exist for the characterization of dynamics for crystalline biomolecules. Solid-state NMR can be used to probe detailed dynamic information in crystalline biomolecules. Recent advances in high-resolution solid-state NMR have enabled the site-specific assignment of (13)C and (15)N nuclei in proteins. With the use of multidimensional separated-local-field experiments, we report the backbone and side chain conformational dynamics of ubiquitin, a globular microcrystalline protein. The measurements of molecular conformational order parameters are based on heteronuclear dipolar couplings, and they are correlated to assigned chemical shifts, to obtain a global perspective on the sub-microsecond dynamics in microcrystalline ubiquitin. A total of 38 Calpha, 35 Cbeta and multiple side chain unique order parameters are collected, and they reveal the high mobility of ubiquitin in the microcrystalline state. In general the side chains show elevated motion in comparison with the backbone sites. The data are compared to solution NMR order parameter measurements on ubiquitin. The SSNMR measurements are sensitive to motions on a broader time scale (low microsecond and faster) than solution NMR measurements (low nanosecond and faster), and the SSNMR order parameters are generally lower than the corresponding solution values. Unlike solution NMR relaxation-based order parameters, order parameters for (13)C(1)H(2) spin systems are readily measured from the powder line shape data. These results illustrate the potential for detailed, extensive, and site-specific dynamic studies of biopolymers by solid-state NMR.

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes α-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge-dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ∼40 kHz, with a total open-state population of ∼20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.