離子對之側鏈長短對β-Hairpin的影響及各種α/β混合胜肽對調鈣素的抑制

Abstract

β-Sheet是一種常見的二級結構，此結構可以藉由股與股間的作用力而得到穩定，例如：主鏈間的氫鍵或側鏈間的靜電作用力。靜電作用力中的離子對作用力可以由兩種不同電性的胺基酸（例如：離胺酸、精胺酸、麩胺酸、天冬胺酸）形成；雖然帶有電性的非自然界胺基酸側鏈長短不同於自然界胺基酸，但仍具有形成離子對的性質，卻不被用於參與蛋白質的組成。因此，我們將用二維核磁共振光譜來研究，在β-hairpin系統中，其胺基酸側鏈長短對於結構穩定度和離子對作用力的影響。殘基α氫原子化學位移，3JNHα藕合常數及NOE效應將用於確認胜肽鏈的β-hairpin結構。β-hairpin結構的的穩定度為，HPTAspLys ~ HPTAspOrn > HPTAspDab；藉由雙突變循環分析，離子對作用力的強度為Asp-Lys ~ Asp-Orn ~ Asp-Dab。 調鈣素是一個高度保守且富含於真核細胞中的蛋白質，而調鈣素參與了多種生理反應，例如：肌肉收縮，胰島素和腦下垂體的分泌，和神經傳遞。調鈣素可以與鹼性兩親性的α-helix胜肽鏈結合。β-胺基酸的主鏈比α-胺基酸胜肽鏈還多一個亞甲基，因此β-胺基酸在三維空間的蛋白質中，可以調整側鏈指向與排列，並且具有抗酵素的水解的特性。在此研究中，我們讓胜肽鏈的側鏈序列相同，改變主鏈的α/β混合樣式（αβ，ααβ，αααβ，和ααβαααβ），透過CD光譜儀和螢光儀，研究不同主鏈α/β混合胜肽鏈對於調鈣素結合力的影響，並且用胰蛋白酶來檢測胜肽鏈的抗水解特性。

β-Sheet is a common secondary structure. The structure can be stabilized by cross strand interactions such as backbone hydrogen bonds and side chain electrostatic interactions. Ion pairing interactions can form between two oppositely charged amino acids, such as Lys, Arg, Glu, and Asp. However, some unnatural amino acids contain different number of methylene groups in the side chain are not incorporated in natural proteins. Accordingly, the ion pairing interaction involving Asp-Lys, Asp-Orn, and AspDab were investigated in a β-hairpin system. The 2D-NMR spectra including TOCSY, ROESY, and COSY were acquired for the peptides. The peptide β-hairpin structure as confirmed by chemical shift deviations, 3JNHα coupling constants, and NOE connectivities. The β-hairpin population and stability were determined by comparing the chemical shifts with the reference peptides. The peptide stability followed the trend HPTAspLys ~ HPTAspOrn > HPTAspDab. The ion pairing interactions were derived from double mutant cycles. The corresponding ion pairing interactions followed the trend Asp-Lys ~ Asp-Orn ~ Asp-Dab. Calmodulin (CaM) is a highly conserved and abundant protein in eukaryotic cells. Calmodulin participates in many physiological processes, such as muscle contraction, insulin and pituitary secretion, and neurotransmission. CaM can bind to basic amphiphilic α-helical peptides. To control the 3D-arrangement of side chains and minimize proteolytic degradation, β-amino acids were incorporated into peptides. β-Amino acids are unnatural amino acids containing one extra methylene group in the backbone compared to natural α-amino acids. In this study, the side chain sequences of peptides were the same as the basic amphiphilic α-helical peptide, but the backbone patterns were varied by mixing α- and β-amino acids, including αβ, ααβ, αααβ, and ααβαααβ. The binding affinities were determined by CD spectroscopy and fluorescence anisotropy. The proteolytic susceptibilities were tested against trypsin.