A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.
A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.
复制标题
蛋白质序列和结构之间关系的测试：脱辅基细胞色素 b5 中血红素结合位点的切除。
DOI：
10.1002/pro.5560070914
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Lecomte,JT
中科院分区：
文献类型：
--
作者：
Constans,AJ;Mayer,MR;Sukits,SF;Lecomte,JT
大鼠肝全细胞色素b5的水溶性结构域是一种αβ蛋白，含有序列β 1-α 1-β4-β3-α2-α3-β5-α4-α5-β2-α6中的二级结构元素。血红素基团由四个螺旋α2、α3、α4和α5包围。为了检验小B血红素蛋白可以由两部分构成的假设，一部分形成血红素位点，另一部分形成组织支架，制备了对应于β 1-α 1-β4-β3-Δ-β2-α6的蛋白片段，其中Δ是绕过血红素结合位点的七个残基接头。通过圆二色谱法发现该片段（“abridged b5”）含有α和β二级结构，通过Trp荧光发射光谱法发现该片段含有三级结构。NMR数据揭示了一种光谱性质与全长脱辅基蛋白相似的物质。这种折叠形式在化学位移时间尺度上与其他较少折叠的物种处于缓慢平衡。通过圆二色谱、吸收和荧光光谱以及尺寸排阻色谱-快速蛋白液相色谱（SEC-FPLC）监测的热变性证实了至少两种不同构象系综的共存。得出的结论是，该蛋白质片段能够采用可能与细胞色素b5相关的特定折叠，但没有实现高的热力学稳定性和协同性。摘要b5表明剪接序列包含折叠蛋白质所必需的信息。这表明，限制链搜索构象空间的主要影响因素是局部水平的结构倾向，而不是内部堆积。序列还具有生成展开屏障所必需的属性。
The water‐soluble domain of rat hepatic holocytochrome b5is anαβ protein containing elements of secondary structure in the sequence βl ‐α l ‐β4‐β3‐α2‐α3‐β5‐α4‐α5‐β2‐α6. The heme group is enclosed by four helices,α2,α3,α4, andα5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to βl‐αl‐β4‐β3‐Δ‐β2‐α6 was prepared, where Δ is a seven‐residue linker bypassing the heme binding site. The fragment (“abridged b5”) was found to containα and β secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full‐length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size‐exclusion chromatography‐fast protein liquid chromatography (SEC‐FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.