Houston M E, Campbell A P, Lix B, Kay C M, Sykes B D, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1996 Aug 6;35(31):10041-50. doi: 10.1021/bi952757m.
A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.
基于序列Ac-EXEALKKEXEALKK-酰胺，制备了一系列内酰胺桥连的和线性的14残基两亲性α-螺旋肽，以确定降低非极性面的疏水性对螺旋含量和稳定性的影响。通过将X位点替换为异亮氨酸、缬氨酸和丙氨酸来实现这一点。在Glu3和Lys7以及Glu10和Lys14的侧链之间形成了间隔为i到i + 4的内酰胺桥，而线性非环化肽可能与相同残基形成i到i + 4的盐桥。通过圆二色光谱法测定发现，在所有情况下，内酰胺桥连肽比相应的线性肽具有更高的螺旋性。此外，对于X = 异亮氨酸和丙氨酸的内酰胺桥连肽，螺旋含量接近100%，对于X = 缬氨酸的情况，螺旋含量大于80%。对于X = 异亮氨酸和缬氨酸，部分原因是内酰胺桥相对于相同序列的线性形式增强链间相互作用的能力。尺寸排阻色谱表明，基于异亮氨酸的肽以二聚体形式缔合。通过浓度依赖性研究和尺寸排阻色谱测定发现，基于丙氨酸的内酰胺桥连肽是单体形式。在良性介质中的热变性研究表明，基于内酰胺的肽非常稳定。通过二维核磁共振光谱进一步表征了基于丙氨酸的内酰胺肽的构象，发现其高度呈螺旋状。结果表明内酰胺桥能够稳定螺旋构象并增强基于3,4疏水七肽重复序列的肽的二聚化。在α-螺旋的疏水面上替换丙氨酸残基可以防止二聚化并确定单体螺旋结构。
