McNulty Joe C, Thompson Darren A, Carrasco Michael R, Millhauser Glenn L
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
FEBS Lett. 2002 Oct 9;529(2-3):243-8. doi: 10.1016/s0014-5793(02)03352-5.
A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10)-helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.
本文描述了一种在化学合成的肽和蛋白质中进行位点定向放置氮氧化物自旋标记的新方法。该方案利用了一种新型二氨基丙酸支架来独立控制主链和侧链的延伸。结果得到了一种自旋标记的侧链，称为Dap-SL，其中酰胺键在氮氧化物和肽主链之间形成连接体。该方法在一系列螺旋肽中得到了验证。圆二色性和核磁共振表明，Dap-SL对螺旋结构仅引入了轻微的扰动。单标记物种的电子顺磁共振光谱允许确定自旋标记的旋转相关时间，并表明Dap-SL侧链比位点定向自旋标记研究中常用的修饰半胱氨酸侧链更灵活。双标记肽的光谱表明存在3(10)-螺旋和α-螺旋的混合物，这与先前研究的结果一致。这里展示的方案为将自旋标记引入蛋白质提供了一种全新的方法，并有望显著扩展对大型蛋白质和受体的生物物理研究。此外，该技术很容易进行修改以纳入任何生物物理探针。
