首页> 外文学位 >BIOPHYSICAL CHARACTERIZATION OF LIPID-PROTEIN AND LIPID-LIPID INTERACTIONS IN MODEL AND RECONSTITUTED MEMBRANES USING STATIC AND REAL-TIME X-RAY DIFFRACTION AND FLUORESCENCE QUENCHING TECHNIQUES.
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BIOPHYSICAL CHARACTERIZATION OF LIPID-PROTEIN AND LIPID-LIPID INTERACTIONS IN MODEL AND RECONSTITUTED MEMBRANES USING STATIC AND REAL-TIME X-RAY DIFFRACTION AND FLUORESCENCE QUENCHING TECHNIQUES.

机译:使用静态和实时X射线衍射和荧光猝灭技术,对模型和重组膜中的脂蛋白和脂脂相互作用进行生物物理表征。

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摘要

(1) The dependence of function and lipid binding affinity of an integral transport protein on the fatty acyl chain characteristics of a membrane-forming phospholipid have been determined. Lipid-protein interactions were examined using a fluorescence quenching technique employing spin-labelled phosphatidylcholine (PC). These studies show that the Ca('2+) ATPase from rabbit sarcoplasmic reticulum binds with equal affinity a variety of PC's used to reconstitute the protein into enzymatically active vesicles, regardless of fatty acyl chain length or details of unsaturation. In parallel with the lipid binding studies, the sensitivity of both catalytic activities of the Ca('2+) ATPase to acyl chain characteristics of the PC membranes in which the enzyme was reconstituted has been measured. The enzyme is sensitive only to the effective acyl chain length, which determines the thickness of the bilayer in which the protein is inserted and displays little sensitivity to such details of unsaturation as degree, position and isomeric type.; (2) To estimate the distribution of a membrane protein between gel and liquid crystal phases in a lipid bilayer using the fluorescence quenching technique, the phase behaviour of the lipids in that membrane must be known. To this end, the phase diagram of hydrated unoriented samples of egg phosphatidic acid (egg PA) with (a) egg PC, (b) dimyristoyl PC and (c) dipalmitoyl PC was determined by the method of x-ray diffraction using synchrotron radiation as the x-ray source. The phase properties of egg PA were unaffected by pH in the range 6-8 or by high concentrations of Na('+), Li('+), K('+), Cs('+), F('-), Cl('-), Br('-) and I('-). Ca('2+), Mg('2+), Sr('2+) and Cd('2+), all induced gel phase formation in the egg PA/egg PC system with Cd('2+) and Sr('2+) effecting the more complete isothermal transformation.; (3) The x-ray diffraction pattern of unoriented hydrated dipalmitoyl PC membranes through the thermotropic gel/liquid crystal phase transition has been observed and recorded in real-time. Synchrotron radiation was used as the x-ray source and the dynamic display of the diffracted image was effected using a 4-stage image intensifier tube coupled to an external fluorescent screen. An upper bound of 2s on the transition has been determined. The general applicability of this newly developed method has been demonstrated in polyethylene and rubber stretching, crystaline polyethylene annealing and clay mineral hydration/dehydration experiments.
机译:(1)已经确定功能性和整体转运蛋白的脂质结合亲和力对成膜磷脂的脂肪酰基链特征的依赖性。使用自旋标记的磷脂酰胆碱(PC)的荧光淬灭技术检查了脂蛋白相互作用。这些研究表明,来自兔肌质网的Ca('2+)ATPase具有相同的亲和力,可与多种PC结合使用,从而将蛋白质重构为酶活性囊泡,而与脂肪酰基链长度或不饱和细节无关。与脂质结合研究并行,已测量了Ca('2+)ATPase的两种催化活性对其中重组有该酶的PC膜的酰基链特征的敏感性。该酶仅对有效的酰基链长度敏感,该长度决定了插入蛋白质的双层的厚度,并且对诸如程度,位置和异构体类型等不饱和细节几乎不敏感。 (2)为了使用荧光猝灭技术估算脂质双层中凝胶相和液晶相之间膜蛋白的分布,必须知道该膜中脂质的相行为。为此,通过x射线衍射法(使用同步加速器辐射)确定了卵磷脂酸(eg PA)与(a)卵PC,(b)二豆蔻酰基PC和(c)二棕榈酰PC的水合未取向样品的相图。作为X射线源。鸡蛋PA的相特性不受pH在6-8范围内或高浓度的Na('+),Li('+),K('+),Cs('+),F('-)的影响,Cl('-),Br('-)和I('-)。 Ca('2 +),Mg('2 +),Sr('2+)和Cd('2+),在Cd('2+)和Sr的鸡蛋PA /鸡蛋PC系统中均诱导凝胶相形成('2+)实现更完全的等温转变。 (3)已经观察到并实时记录了未取向的水合二棕榈酰PC膜通过热致凝胶/液晶相变的x射线衍射图。同步辐射用作X射线源,使用耦合到外部荧光屏的4级图像增强管实现衍射图像的动态显示。确定了过渡的2s上限。这种新开发的方法的普遍适用性已在聚乙烯和橡胶拉伸,结晶聚乙烯退火和粘土矿物水化/脱水实验中得到证明。

著录项

  • 作者

    CAFFREY, MARTIN DENIS.;

  • 作者单位

    Cornell University.;

  • 授予单位 Cornell University.;
  • 学科 Biophysics General.
  • 学位 Ph.D.
  • 年度 1982
  • 页码 214 p.
  • 总页数 214
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;
  • 关键词

  • 入库时间 2022-08-17 11:51:29

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