Stanley Will A, Versluis Kees, Schultz Carsten, Heck Albert J R, Wilmanns Matthias
EMBL-Hamburg, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.
Arch Biochem Biophys. 2007 May 1;461(1):50-8. doi: 10.1016/j.abb.2007.02.026. Epub 2007 Mar 15.
Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.
在长链脂肪酰辅酶A（LCFA-CoAs）和具有等效脂肪酸链长度的肉碱衍生物（LCFA-肉碱）存在的情况下，通过近天然电喷雾电离质谱法对固醇载体蛋白2（SCP2）进行了研究。比较了四种SCP2构建体，以研究N端前序列和C端过氧化物酶体靶向信号对配体结合的影响。去除N端或C端残基不影响配体结合。LCFA-CoAs是SCP2的高亲和力配体这一观察结果得到了证实，而LCFA-肉碱首次被证明不与SCP2相互作用。LCFA-CoAs与SCP2形成化学计量比为2:1和1:1的非共价复合物，在离子进入质谱仪时通过提高离子能量可使其解离。使用尼罗红丁酸的荧光竞争测定法证实了溶液中的质谱观察结果。讨论了SCP2缺乏LCFA-肉碱结合的生理意义。
