Study of hemagglutinin membrane fusion domain

流感病毒糖蛋白血凝素（HA） HA2结构域的23个n端残基中，除5个外，在所有16种血清型HA基因中都是严格保守的。这种HA2融合肽（HAfp）的结构和功能一直是广泛的生物物理、计算和功能分析的焦点，但大多数这些分析都是不包括严格保守残基Trp21-Tyr22-Gly23的肽。我们对溶解在十二烷基磷脂酰胆碱（DPC）中的血清型H1亚型的全长HAfp进行了异核三重共振NMR研究，先前发现其具有非常紧密的螺旋发夹结构，其n端α -螺旋（Gly1-Glu11）与其第二个α -螺旋（Trp14-Gly23）紧密排列，七个保守的甘氨酸残基中有六个位于螺旋界面。位于第13位的第七个保守残基采用正的phi角，使得连接两个螺旋的发夹旋转成为可能。发现该结构被多个螺旋间的CaH - C=O氢键所稳定，以强螺旋间的HN-Ha为特征；和Ha-Hb NOE接触。新的证据表明，这种发夹结构有一个额外的稳定力，即n端Gly1氨基和螺旋2的偶极矩之间的强电荷偶极相互作用。利用一种新型的基于亚甲基trosy的三维核磁共振实验对氨基端15N共振进行pH滴定，并观察到Gly1 13C'的pK值为8.8，大大高于亲脂环境中n端氨基的预期值。螺旋2的三个c端羰基碳的化学位移与Gly1-N的质子化状态相匹配，表明n端氨基与螺旋2的轴之间非常接近，从而为反平行发夹折叠提供了最佳的电荷偶极子稳定性。Glu11和Asp19侧链羧酸基团的pK值分别比水溶性蛋白质中溶剂暴露侧链的pK值高约1和0.5个单位，表明介电常数为epsilon&#61472；= 30 （Glu11）和epsilon&#61472；= 60 (Asp19)，这将这些基团置于磷脂胶束的头基区域。

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 Trp21-Tyr22-Gly23. Our heteronuclear triple resonance NMR study of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline (DPC), previously revealed a remarkably tight helical hairpin structure, with its N-terminal alpha-helix (Gly1-Glu11) packed tightly against its second alpha-helix (Trp14-Gly23), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive phi angle, enabling the hairpin turn that links the two helices. The structure was found to be stabilized by multiple interhelical CaH to C=O hydrogen bonds, characterized by strong interhelical HN-Ha; and Ha-Hb NOE contacts. New evidence for an additional stabilizing force of this hairpin structure has now been identified, namely a strong charge dipole interaction between the N-terminal Gly1 amino group and the dipole moment of helix 2. pH titration of the amino-terminal 15N resonance, using a novel methylene-TROSY based 3D NMR experiment, and observation of Gly1 13C' show a strongly elevated pK value of 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly1-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, thereby providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side chain carboxylate groups of Glu11 and Asp19 are higher by about one and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of epsilon = 30 (Glu11) and epsilon= 60 (Asp19), which places these groups in the headgroup region of the phospholipid micelle. Biological membranes present a highly fluid environment and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane, and rotationally about the normal vector of this plane. We have found that whole-body motions of proteins within a lipid bilayer can be determined from NMR 15N relaxation rates collected for different size bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we find large-amplitude, rigid-body wobbling motions on the nanosecond timescale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematically varying the size of one is applicable to a wide range of systems beyond protein-membrane interactions.