Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy.
Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy.
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通过反相 HPLC、CD 和 NMR 光谱检测催乳素受体富含脯氨酸基序 (PRM) 肽的多个顺反构象异构体。
DOI：
10.1042/bj3150833
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Shearer,WT
中科院分区：
文献类型：
--
作者：
O'Neal,KD;Chari,MV;Mcdonald,CH;Cook,RG;Yu-Lee,LY;Morrisett,JD;Shearer,WT
用一维和二维质子核磁共振波谱研究了一个由8个氨基酸组成的合成肽（Ile 1-Phe 2-Pro3-Pro4-Val 5-Pro6-Gly 7-Pro 8）在水和DMSO中的构象动力学。纯化的PRL-R PRM肽在20 °C下在反相HPLC（RP-HPLC）上以60：40的比例洗脱为平衡的两个部分分辨的峰。在30 °C下，两个峰合并成单个峰。两个RP-HPLC峰对应于由四个脯氨酸酰胺键之一的缓慢顺式-转异构化产生的两种肽构象异构体。虽然该肽只有三个酰胺（NH）质子，但其在水中的1D NMR光谱包含约15个可辨别的NH区域峰，为多种构象异构体提供了证据。根据2D-COSY谱、化学位移值、共振分裂模式和温度系数对酰胺的共振峰进行了归属。根据适当共振的积分强度和/或峰高计算，每种脯氨酸在水中的反式比为Phe 2-Pro 3（35：65）、Pro 3-Pro 4（40：60）、Val 5-Pro 6（70：30）和Gly 7-Pro 8（30：70）。温度研究（25-70 °C）用于半定量地估计不同脯氨酸的异构化速率。在水中，Pro 8经历快速异构化; Pro3以中等速率异构化;而Pro4和Pro6似乎都非常缓慢地异构化，因为没有观察到酰胺共振的聚结。在DMSO中，只有Pro 4表现出缓慢的异构化。缓慢的动力学结合通过RP-HPLC和NMR测定的相似的60：40的构象异构体比例表明，Pro3-Pro4键的异构化产生了两个RP-HPLC峰。在NMR实验中观察到近端和远端脯氨酸异构化效应。所有的16个理论（24= 16）脯氨酸的配置似乎存在于水中的平衡。主要（19%）的构象，trans 3-trans 4-cis 6-trans 8，可能反映了在天然PRL-R的PRM脯氨酸的配置。Pro6从顺反异构化的肽N-和C-末端之间的相互作用，表明一个整体的假环状构象。这种全反式脯氨酸构型可能在细胞因子/促红细胞生成素受体的功能中发挥重要的生化作用。提出了一个模型，这表明异构化的PRM亲免蛋白，如FK 506结合蛋白（FKBP）作为一个开关开关细胞因子受体激活。
An eight-amino-acid synthetic peptide (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 °C with a ratio of 60:40. At 30 °C, the two peaks coalesced into a single peak. The two RP-HPLC peaks correspond to two peptide conformers resulting from the slowcis–transisomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its 1D NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values, resonance splitting patterns and temperature coefficients. Thecis:transratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3(35:65), Pro3-Pro4(40:60), Val5-Pro6(70:30), and Gly7-Pro8(30:70). Temperature studies (25–70 °C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8undergoes rapid isomerization; Pro3isomerizes at an intermediate rate; while Pro4and Pro6both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24= 16) proline configurations appear to exist in equilibrium in water. The predominant (19%) conformation,trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6fromcistotransgenerates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-transproline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK506-binding protein (FKBP) serves as an on–off switch for cytokine receptor activation.