通过戊二醛对游离胺进行还原环戊基化实现五肽重复蛋白的结晶。
Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde.
作者信息
Vetting Matthew W, Hegde Subray S, Blanchard John S
机构信息
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
出版信息
Acta Crystallogr D Biol Crystallogr. 2009 May;65(Pt 5):462-9. doi: 10.1107/S0907444909008324. Epub 2009 Apr 18.
Abstract
The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane-dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 A resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.
摘要
粪肠球菌的五肽重复蛋白EfsQnr可保护DNA回旋酶免受氟喹诺酮类药物的抑制。EfsQnr被克隆并纯化至同质，但在初始结晶筛选中未能产生适合衍射的晶体。用戊二醛和强还原剂硼烷二甲胺处理EfsQnr，得到一种衍生化蛋白，该蛋白产生的晶体衍射分辨率达到1.6 Å；随后通过单波长反常色散确定了其结构。使用傅里叶变换离子回旋共振质谱对衍生化蛋白进行分析表明，每个游离氨基的质量增加了68 Da。关于有限数量结构有序赖氨酸的电子密度图表明，修饰是游离胺的环状戊基化，产生哌啶基团。
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