Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.
Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.
复制标题
通过自旋标记 EPR 和频域荧光光谱解析模型膜中的磷脂构象异质性。
DOI：
10.1016/s0006-3495(91)82281-0
复制
发表时间：
1991
影响因子：
3.4
通讯作者：
Lakowicz,JR
中科院分区：
文献类型：
--
作者：
Squier,TC;Mahaney,JE;Yin,JJ;Lai,CS;Lakowicz,JR
我们利用硬脂酸的荧光和氮氧衍生物作为磷脂囊泡在生理条件下膜结构异质性的探针，以及不同离子强度和温度的条件下，光谱异质性已经被观察到，并归因于探针的多种电离状态。为了确定这种光谱异质性的来源，我们利用了(a)硬脂酸的自旋标记和邻氧基衍生物（即SASL和AS）和(b)磷脂酰胆碱的二苯基六烯衍生物（DPH-PC）在含有二肉豆烯磷脂酰胆碱（DMPC）的单组分膜中的弛弛性（寿命）和运动的互补测量。我们使用15N硬脂酸自旋标签对膜非均质性的最佳敏感性。将荧光磷脂类似物DPH-PC（在该pH范围内没有可电离基团）的寿命和动力学与AS的寿命和动力学进行比较，使我们能够区分膜结构的变化和标签的电离。DPH-PC的量子产率和旋转动力学与pH无关，表明pH的变化不影响宿主磷脂的构象。然而，SASL的EPR光谱和AS的寿命或动力学都受到溶液pH变化的深刻影响。这两种探针在DMPC膜中的表观pKa测定值接近pH 6.3，这意味着在生理pH和离子强度下，这些硬脂酸标签主要以单一电离群体的形式存在于膜中。因此，观察到的SASL光谱的温度和离子强度依赖性变化以及中性pH下DMPC膜中as的寿命或动力学是由于膜结构的变化而不是探针的电离。离子梯度跨越生物膜诱导磷脂结构改变的可能性，从而调节脂质-蛋白相互作用进行了讨论。
We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.