Schreier Shirley, Barbosa Simone R, Casallanovo Fábio, Vieira Renata de F F, Cilli Eduardo M, Paiva Antonio C M, Nakaie Clóvis R
Laboratory of Structural Biology, Department of Biochemistry, Institute of Chemistry, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
Biopolymers. 2004 Aug 5;74(5):389-402. doi: 10.1002/bip.20092.
N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.
合成了含有顺磁性氨基酸2,2,6,6-四甲基哌啶-1-氧基-4-氨基-4-羧酸（TOAC）的血管紧张素（AII，DRVYIHPF）和缓激肽（BK，RPPGFSPFR）的N端和内部标记类似物。TOAC取代了AII中的Asp1（TOAC1-AII）和Val3（TOAC3-AII），并插入到BK的Arg1之前（TOAC0-BK）以及取代Pro3（TOAC3-BK）。研究了肽的构象性质与三氟乙醇（TFE）含量和pH值的关系。电子顺磁共振光谱对这两个变量都很敏感，结果表明，内部标记的类似物产生的旋转相关时间（tauC）比N端标记的类似物大得多，这表明N端的运动自由度更大。在TFE中，由于粘度效应，tauC增加。tau（C肽）/tau（CTOAC）比值的计算表明，肽获得了更多折叠构象。圆二色光谱表明，除了TFE中的TOAC1-AII外，N端标记的类似物表现出与母体肽相似的构象行为。相比之下，在所有条件下，TOAC3衍生物获得的构象更为受限。AII及其衍生物的荧光光谱对Tyr4的电离特别敏感。对于TOAC3-AII，氮氧化物部分的荧光猝灭更为明显。TOAC衍生物的构象行为与其生物活性具有良好的相关性，因为虽然N端标记的肽具有部分活性，但其内部标记的对应物无活性[中井，C.R.等人，《肽》2002年，23卷，65 - 70页]。数据表明，在肽链中间插入TOAC会引起构象限制，导致主链柔韧性丧失，使肽无法获得其与受体结合的构象。
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