Tuning the selectivity of protein photocleavage: Spectroscopic and photochemical studies
Tuning the selectivity of protein photocleavage: Spectroscopic and photochemical studies
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DOI：
10.1021/ja9844377
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发表时间：
1999-05-05
影响因子：
15
通讯作者：
Buranaprapuk, A
中科院分区：
文献类型：
--
作者：
Kumar, CV;Buranaprapuk, A
N-4-(1-芘)丁酰-L-苯丙氨酸(Py-Phe)对溶菌酶的定点光裂解促使我们研究了肽基侧链在决定蛋白质光裂解特异性中的作用。本文描述了Py-Gly、Py-(Gly)(N)-Phe(n=0，1，2)和Py-Phe-Gly-Gly的光谱和光切割性质。这些分子的连接基长度/结构对探针的光谱和光切割性质有深远的影响。随着探针Py-(Gly)(N)-Phe与蛋白质结合，其吸收光谱的变化与连接物的结构或长度无关。这些探针与牛血清白蛋白(BSA)或溶菌酶等蛋白质的结合常数在2.2之间变化。+/-0.3×10(5)dm(3)摩尔(-1)至6.5+/-0.4 x 10(7)dm(3)摩尔(-1)。Py-Phe和Py-Phe-Gly-Gly与BSA的结合常数比Py-Gly、Py-Gly-Phe或Py-Gly-Gly-Phe大一个数量级。与吸收变化相反，荧光光谱的变化取决于探针结构，这表明连接物结构对探针结合性质有微妙的作用。Py-Phe和Py-Phe-Gly与牛血清白蛋白结合时，除了Py-Phe和Py-Phe-Gly-Gly外，在466 nm处还观察到了新的宽广的发射，而其他探针或这些探针中的任何一个与溶菌酶结合时都没有观察到这样的发射。从圆二色谱(CD)可以确定探针之间更清晰的区别。没有两个探针-蛋白质络合物的CD光谱是重叠的，清楚地表明了蛋白质基质中吡喃基发色团周围的手性环境的差异。以钴(III)-六亚胺(COHA)为猝灭剂的荧光猝灭实验表明，与BSA结合的荧光团具有广泛的保护作用，而与溶菌酶结合的发色团相对容易获得。三肽探针比二肽探针具有更好的保护作用。这些探针中的羧基和吡喃基之间的分离距离对探针对COHA的可达性有很大的影响。这些差异反过来预计会影响光切割效率。在COHA作为电子受体的情况下，在344 nm处照射探针-蛋白质复合体，观察到溶菌酶的光解。所有的探针对溶菌酶都表现出高度的特异性，并且只有两条产物带。对于BSA，蛋白质切割位点的位置和特异性随着探针结构的不同而变化很大，这表明作为探针结构的函数，选择性发生了重大变化。对溶菌酶的光片段进行了肽序列分析，揭示了光裂解位点的位置。当Py-(Gly)，-Phe(n=1，2)在Trp108和Val109之间切割溶菌酶时，Py-(Gly)(N)-Phe(0，1，2)和Py-Phe-Gly-Gly在Ala110/Trp111处观察到第二个微小的切割位点。含有溶菌酶的主要产物和次要产物的产率之比取决于连接体的性质。在没有探针、COHA或光的情况下，没有观察到蛋白质切割。光谱和光化学研究表明，探针的结合和切割特异性随侧链结构的不同而不同。
Site-specific photocleavage of lysozyme by N-4( 1-pyrene)butyroyl-L-phenylalanine (Py-Phe) prompted us to investigate the role of the peptidyl side chain in determining the specificity of the protein photocleavage. The spectroscopic and photocleavage properties of Py-Gly, Py-(Gly)(n)-Phe (n = 0, 1, 2), and Py-Phe-Gly-Gly, in this context, is described here. The linker length/structure of these molecules has a profound effect on the spectroscopic and photocleavage properties of the probes. The absorption spectral changes accompanying the binding of the probes Py-(Gly)(n)-Phe to the proteins were independent of the linker structure or length. Binding constants of these probes with proteins such as bovine serum albumin (BSA) or lysozyme, varied from 2.2. +/- 0.3 x 10(5) dm(3)mol(-1) to 6.5 +/- 0.4 x 10(7) dm(3)mol(-1). Binding constants of Py-Phe and Py-Phe-Gly-Gly for BSA have been an order of magnitude larger than those of Py-Gly, Py-Gly-Phe or Py-Gly-Gly-Phe. The fluorescence spectral changes, in contrast to the absorption changes, depended on the probe structure suggesting the subtle role of the linker structure on the probe binding properties. In addition to the pyrenyl fluorescence, new, broad emission was observed at 466 nm with Py-Phe and Py-Phe-Gly-Gly when bound to BSA, but no such emission was observed with the other probes or with any of these probes bound to lysozyme. Much clearer distinction between the probes can be ascertained from the circular dichroism (CD) spectra. No two CD spectra of the probe-protein complexes were superimposable, clearly demonstrating the differences in the chiral environment surrounding the pyrenyl chromophore in the protein matrix. Fluorescence quenching experiments using Co(III)hexammine (CoHA) as the quencher indicate extensive protection of the fluorophore bound to BSA, while the chromophore bound to lysozyme was relatively more accessible. The tripeptide probes were protected better than the dipeptide probes. The distance of separation between the carboxyl function and the pyrenyl group in these probes has a substantial effect on the accessibility of the probe to CoHA. These differences are, in turn, expected to influence the photocleavage efficiencies. Photocleavage of lysozyme was observed when probe-protein complexes have been irradiated at 344 nm in the presence of CoHA as an electron acceptor. All of the probes showed high specificity with lysozyme and resulted in just two product bands. In case of BSA, the protein cleavage site location and specificity varied drastically with the probe structure, suggesting major changes in the selectivity as a function of probe structure. Peptide sequencing studies of the photofragments from lysozyme revealed the location of the photocleavage sites. While Py-(Gly),-Phe (n = 1, 2) cleave lysozyme at a site between Trp108 and Val109, similar to that for Py-Phe, a second minor cleavage site at Ala110/Trp111 was observed for Py-(Gly)(n)-Phe (0, 1, 2) and Py-Phe-Gly-Gly. The ratio of the yields of the major to the minor product, with lysozyme, depended on the nature of the linker. No protein cleavage was observed in the absence of the probe or CoHA or light. The spectral and the photochemical studies indicate the binding and cleavage specificity of the probes vary with the side chain structure.