Bodner Michele L, Gabrys Charles M, Parkanzky Paul D, Yang Jun, Duskin Craig A, Weliky David P
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Magn Reson Chem. 2004 Feb;42(2):187-94. doi: 10.1002/mrc.1331.
HIV-1 and influenza viral fusion peptides are biologically relevant model fusion systems and, in this study, their membrane-associated structures were probed by solid-state NMR (13)C chemical shift measurements. The influenza peptide IFP-L2CF3N contained a (13)C carbonyl label at Leu-2 and a (15)N label at Phe-3 while the HIV-1 peptide HFP-UF8L9G10 was uniformly (13)C and (15)N labeled at Phe-8, Leu-9 and Gly-10. The membrane composition of the IFP-L2CF3N sample was POPC-POPG (4:1) and the membrane composition of the HFP-UF8L9G10 sample was a mixture of lipids and cholesterol which approximately reflects the lipid headgroup and cholesterol composition of host cells of the HIV-1 virus. In one-dimensional magic angle spinning spectra, labeled backbone (13)C were selectively observed using a REDOR filter of the (13)C-(15)N dipolar coupling. Backbone chemical shifts were very similar at -50 and 20 degrees C, which suggests that low temperature does not appreciably change the peptide structure. Relative to -50 degrees C, the 20 degrees C spectra had narrower signals with lower integrated intensity, which is consistent with greater motion at the higher temperature. The Leu-2 chemical shift in the IFP-L2CF3N sample correlates with a helical structure at this residue and is consistent with detection of helical structure by other biophysical techniques. Two-dimensional (13)C-(13)C correlation spectra were obtained for the HFP-UF8L9G10 sample and were used to assign the chemical shifts of all of the (13)C labels in the peptide. Secondary shift analysis was consistent with a beta-strand structure over these three residues. The high signal-to-noise ratio of the 2D spectra suggests that membrane-associated fusion peptides with longer sequences of labeled amino acids can also be assigned with 2D and 3D methods.
HIV-1和流感病毒融合肽是具有生物学相关性的模型融合系统，在本研究中，通过固态核磁共振（13）C化学位移测量来探测它们的膜相关结构。流感肽IFP-L2CF3N在Leu-2处含有一个（13）C羰基标记，在Phe-3处含有一个（15）N标记，而HIV-1肽HFP-UF8L9G10在Phe-8、Leu-9和Gly-10处均匀地进行了（13）C和（15）N标记。IFP-L2CF3N样品的膜组成是POPC-POPG（4:1），HFP-UF8L9G10样品的膜组成是脂质和胆固醇的混合物，这大致反映了HIV-1病毒宿主细胞的脂质头部基团和胆固醇组成。在一维魔角旋转光谱中，使用（13）C-（15）N偶极耦合的REDOR滤波器选择性地观察标记的主链（13）C。在-50和20摄氏度时，主链化学位移非常相似，这表明低温不会明显改变肽的结构。相对于-50摄氏度，20摄氏度的光谱信号更窄，积分强度更低，这与较高温度下更大的运动性一致。IFP-L2CF3N样品中Leu-2的化学位移与该残基处的螺旋结构相关，并且与通过其他生物物理技术检测到的螺旋结构一致。获得了HFP-UF8L9G10样品的二维（13）C-（13）C相关光谱，并用于确定肽中所有（13）C标记的化学位移。二级位移分析与这三个残基上的β-链结构一致。二维光谱的高信噪比表明，具有更长标记氨基酸序列的膜相关融合肽也可以通过二维和三维方法进行归属。
