Mani Rajeswari, Waring Alan J, Hong Mei
Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Chembiochem. 2007 Oct 15;8(15):1877-84. doi: 10.1002/cbic.200700335.
Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.
二硫键连接的β-发夹结构在抗菌肽中很常见。已知二硫键对抗菌活性很重要，但其潜在的结构原因尚不清楚。我们研究了抗菌肽protegrin-1的二硫键缺失类似物的膜结合结构，其中四个半胱氨酸残基被丙氨酸取代。通过魔角旋转核磁共振光谱法确定了该丙氨酸-PG1肽在膜中的二级结构、动力学和拓扑结构。从一维交叉极化和直接极化光谱以及二维J耦合介导的碳-碳相关光谱中获得了多个碳-13标记残基的构象依赖性碳-13各向同性化学位移。大多数标记残基呈现两种构象：无规卷曲和β-折叠结构。这种双构象特性在模拟细菌膜的阴离子脂质双层和模拟真核细胞膜的含两性离子胆固醇的双层中均存在。通过二维碳-氢偶极位移相关实验测量了肽的流动性。无规卷曲部分流动性很高，而β-折叠部分则很刚性。从脂质链到肽的氢自旋扩散表明，β-折叠部分很好地插入了阴离子膜中，但在含胆固醇的中性膜中则结合在表面。因此，二硫键的去除使一些PG-1分子变成了与脂质膜结合不良的高流动性无规卷曲，但其他分子保留了β-折叠构象，并且具有与亲本肽相似的膜结合拓扑结构。因此，丙氨酸-PG1抗菌活性降低主要是由于具有插入能力的β-折叠分子数量减少，而不是结构相同的肽的活性普遍减弱。
