Zheng C, Cahill S, Breslow E
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
Biochemistry. 1996 Sep 10;35(36):11763-72. doi: 10.1021/bi960906a.
The active site of liganded neurophysin contains a salt bridge that involves the side chains of Arg-8 and Glu-47 of the protein and the alpha-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or to which the environment of Arg-8 in the unliganded state differs in monomers and dimers, is relevant to an understanding of allosteric mechanism in this system. In the present study, the behavior of the salt bridge was investigated by 15N NMR and chemical replacement of Arg-8. Bovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by 15N-substituted Arg or by other residues using chemical semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord with the view of the salt bridge in the liganded state obtained from crystals of bovine neurophysin-II complexes. In the unliganded state, comparison of the 15N and proton NMR signals from Arg-8 with those in smaller arginine systems suggested the absence of significant interactions between the guanidinium of Arg-8 and Glu-47 or between the amino terminal region of Arg-8 and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and dimers was found. Marked spectral changes accompanying the binding of oxytocin indicated changes in the environment of both the side chain and amino terminal region of Arg-8. The NMR results were in good agreement with a recently emerging comparison of bovine neurophysin-II derivatives in the liganded and unliganded states, with the notable exception of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, significant differences between the two bovine neurophysins in the susceptibility to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential allosteric mechanisms underlying neurophysin ligand-facilitated dimerization.
结合配体的神经垂体激素运载蛋白的活性位点包含一个盐桥，该盐桥涉及蛋白质的精氨酸 -8和谷氨酸 -47的侧链以及结合激素或相关肽的α-氨基。在没有肽的情况下，精氨酸 -8-谷氨酸 -47盐桥持续存在的程度，或者在未结合配体状态下精氨酸 -8的环境在单体和二聚体中的差异程度，对于理解该系统中的变构机制至关重要。在本研究中，通过15N核磁共振和精氨酸 -8的化学置换研究了盐桥的行为。牛神经垂体激素运载蛋白-I被转化为其去1-8衍生物，并且使用化学半合成法将精氨酸 -8替换为15N取代的精氨酸或其他残基。不同氨基酸恢复去1-8蛋白对肽亲和力的相对能力与从牛神经垂体激素运载蛋白-II复合物晶体获得的结合配体状态下盐桥的观点高度一致。在未结合配体的状态下，将精氨酸 -8的15N和质子核磁共振信号与较小精氨酸系统中的信号进行比较表明，精氨酸 -8的胍基与谷氨酸 -47之间或精氨酸 -8的氨基末端区域与蛋白质的其他元件之间不存在明显的相互作用。未发现未结合配体的单体和二聚体之间精氨酸 -8环境存在差异的证据。伴随催产素结合的明显光谱变化表明精氨酸 -8的侧链和氨基末端区域的环境发生了变化。核磁共振结果与最近出现的牛神经垂体激素运载蛋白-II衍生物在结合配体和未结合配体状态下的比较结果高度一致，但未结合配体状态下盐桥形成的程度除外。结果表明与未结合配体状态下在精氨酸 -8处对胰蛋白酶切割的敏感性以及肽结合的pH依赖性方面两种牛神经垂体激素之间的显著差异一致，并有助于解释这些差异，此外还限制了神经垂体激素运载蛋白配体促进二聚化的潜在变构机制。
