Carpenter K A, Wilkes B C, Schiller P W
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Quebec, Canada.
Biopolymers. 1995 Dec;36(6):735-49. doi: 10.1002/bip.360360607.
In the present paper we investigate the influence of sample pH on the conformational and dynamical properties of the pseudotripeptide H-Tyr-Tic psi [CH2-NH]Phe-OH (TIP[psi]; Tic: 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid) using various one- and two-dimensional nmr techniques in conjunction with molecular modeling. Studies were conducted at three different pH levels corresponding to the zwitterionic peptide containing a formal positive charge (pH 3.1), the deprotonated molecule (pH 9.1), and a situation at neutral pH (pH 7.2) involving both protonated and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional1H-nmr spectra reveals that in solution TIP[psi] is in slow dynamic exchange between conformations containing cis and trans configurations of the Tyr-Tic bond. An nmr pH dependence study of the cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bone amine. From the nmr data, reduced peptide bond pK alpha values of 6.5 and 7.5 were determined for the cis and trans conformers, respectively. It was concluded that conformations containing a trans Tyr-Tic bond are stabilized at low pH by an intramolecular hydrogen bond between the Tyr carbonyl and the reduced peptide bond protonated amine. This observation was corroborated by molecular mechanics investigations that revealed low energy trans structures compatible with nmr structural data, and furthermore, were consistently characterized by the existence of a strong N+ H ... O = C interaction closing a seven-membered cycle. The dynamics of cis-trans isomerization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carried out at several temperatures between 50 and 70 degrees C allowed the determination of isomerization rate constants as well as thermodynamic activation parameters. delta G not equal to values were in close agreement with the cis-->trans energy barrier found in X-Pro peptide fragments (approximately 83 kJ/mol). A large entropic barrier determined for the trans-->cis conversion of TIP[psi] (5.7 JK-1 mol-1 at pH 3.1;6.5 J K-1 mol-1 at pH 9.1) is discussed in terms of decreased solvent molecular ordering around the conformers possessing a trans Tyr-Tic bond. Evidence that the neutral form of the reduced peptide bond gains rigidity upon protonation was obtained from relaxation measurements in the rotating frame. T1 rho measurements of several protons in the vicinity of the reduced peptide bond were made as a function of spin-lock field.(ABSTRACT TRUNCATED AT 400 WORDS)
在本论文中，我们使用各种一维和二维核磁共振技术并结合分子建模，研究了样品pH值对假三肽H-Tyr-Tic ψ[CH₂-NH]Phe-OH（TIP[ψ]；Tic：1,2,3,4-四氢异喹啉-3-羧酸）的构象和动力学性质的影响。研究在三个不同的pH水平下进行，分别对应含有形式正电荷的两性离子肽（pH 3.1）、去质子化分子（pH 9.1）以及中性pH（pH 7.2）的情况，其中涉及还原肽键的质子化和去质子化状态。对一维¹H-核磁共振谱的分析表明，在溶液中TIP[ψ]在含有Tyr-Tic键顺式和反式构型的构象之间进行缓慢的动态交换。对顺式:反式比例的核磁共振pH依赖性研究表明，交换过程受还原骨胺的质子化状态控制。根据核磁共振数据，分别确定了顺式和反式构象体的还原肽键pKα值为6.5和7.5。得出的结论是，含有反式Tyr-Tic键的构象在低pH下通过Tyr羰基与还原肽键质子化胺之间的分子内氢键而稳定。分子力学研究证实了这一观察结果，该研究揭示了与核磁共振结构数据兼容的低能量反式结构，此外，其特征还始终表现为存在一个闭合七元环的强N⁺H...O = C相互作用。通过核磁共振交换实验探测了围绕Tyr-Tic肽键的顺反异构化动力学。在50至70摄氏度之间的几个温度下对交换共振进行选择性预饱和，从而确定了异构化速率常数以及热力学活化参数。ΔG≠值与在X-Pro肽片段中发现的顺式→反式能垒（约83 kJ/mol）密切一致。针对TIP[ψ]的反式→顺式转化确定的大熵垒（在pH 3.1时为5.7 J K⁻¹ mol⁻¹；在pH 9.1时为6.5 J K⁻¹ mol⁻¹），根据围绕具有反式Tyr-Tic键的构象体周围溶剂分子有序性的降低进行了讨论。从旋转坐标系中的弛豫测量获得了还原肽键中性形式在质子化时变得刚性增强的证据。对还原肽键附近几个质子的T1ρ测量是作为自旋锁定场的函数进行的。（摘要截取自400字）
