第2章细胞膜和跨膜运输

Polarity:
Molecular size:
Binding specificity:
Gene cloning into expression vector Transformation and Expression Affinity-tag and desalting purification

Provided by an atomic force microscope

In the nonactivated state, lipid rafts float freely, carrying a few passenger proteins. But when activated, they coalesce to form larger platforms where proteins meet to perform functions in signaling, processing, and transport. Clustering of rafts is key to understanding raft function in human physiology as well as in development of human disease (opens an exciting new field in molecular medicine).
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Lipid rafts model
—Lipid rafts are small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes.
• In 1985, Johann Deisenhofer and his colleagues succeeded in solving the first atomicresolution structure of a membrane protein by X-ray crystallography—the bacterial photosynthetic reaction center ( three subunits containing 11 membrane- spanning α helices) • The Nobel Prize for Chemistry in 1988
Lipid raft: 脂筏
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The chemical composition of membranes
— Membrane lipid and membrane protein
Membrane lipid :
• •
— The fluid part of the model
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Membrane lipids are amphipathic There are three major classes of lipids: Phospholipids (Phosphoglyceride-甘油磷 脂 and sphingolipids-鞘脂类); Glycolipids; Sterols (is only found in animals) Fluidity: four kinds of movement of membrane lipids Asymmetry
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The lateral diffusion of membrane lipids can demonstrated experimentally by a technique called fluorescence recovery after photobleaching (FRAP, 荧光漂白恢复)
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The relative size and shape of mitochondrial Complex II–IV, as well as a schematic of Complex I
• The structures of Complex III (cytochrome bc1complex) : Iwata et al., 1998; Lange and Hunte, 2002; Xia et al., 1997; Zhang et al., 1998. • Complex IV (cyto-chrome c oxidase complex): Tsukihara et al., 1995; Tsukihara et al., 1996 • Complex V (ATPase): Abra-anhams et al., 1994. • Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, Bratlam M, Rao Z, Crystal structure of mitochondrial respiratory membrane protein complex II, Cell. 2005; 121(7):1043-57. • The structures of the membrane domain of respiratory complex I from Escherichia coli, and of the entire complex I fromThermus thermophilus (温泉菌-嗜热菌) have been determined. Nature. 2010 May 25 27; 465(7297):441-5.

Fluidity of membrane lipids
• • • •
Laternal diffusion Flexion(屈曲) Rotation Flip-flop(翻转)

The length of the fatty acid The degree of unsaturation of their side chains The temperature The effects of sterols on membrane fluidity
The cell surface is coated with sugar residues — cell coat or glycocalyx (糖萼)
• •
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Membrane proteins are oriented asymmetrically across the lipid bilayer
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Why are proteins the perfect molecule to build structures in the cell membrane?
Functions of membrane protein
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The functions of the cell membranes
Membrane proteins: — The “mosaic” part of the model
Classification
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Integral membrane proteins—amphipathic Peripheral membrane proteins Lipid-anchored proteins
Membrane proteins constitute more than a third of all known proteins Almost 200 unique membrane-protein structures have been revealed Nature Insight (Nature, 2009,459: 343-385)
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细胞中的物质流 第二章 细胞膜和跨膜运输
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Outline
Cell
membrane Membrane transport
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What are some substances commonly transported across membranes?
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• •
Cell membrane or plasma membrane Endomembrane systems Biomembrane
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Classification
甘油磷脂
鞘脂
鞘磷脂
半乳糖神经酰胺
PtdCho – phosphatidylcholine; PtdEtn – phosphatidylethanolamine; PtdIns – phosphatidylinositol; 9 PtdSer – phosphatidylserine. GSL- galactosylceramide,半乳糖神经酰胺
The use of cell fusion to reveal mobility of membrane proteins
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Many membrane proteins are glycosylated
Glycoprotein (糖蛋白：由短的寡糖链与蛋白质共价相连构成的分子) Proteoglycan (蛋白聚糖：蛋白质和糖胺聚糖通过共价键连接所构成 的复合糖)
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Protein purification
Characteristic
Solubility: Ionic charge:
Procedure
1. 2. 1. 2. 3. 1. 2. 3. 4. 1. 2. 3. 4. 1. Salting in(盐溶) Salting out(盐析) Ion exchange chromatography Electrophoresis Isoelectric focusing(等电聚焦) Adsorption chromatography(吸附层析) Paper chromatography Reverse-phase chromatography(反相色谱) Hydrophobic interaction chromatography (疏水层析) Dialysis(透析) Gel electrophoresis Gel filtration chromatography Ultracentrifugation Affinity chromatography
pQE series
Histidine-Tag (metal chelating) affinity chromatography
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Purification to homogeneity checked by electrophoresis
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• •
X-ray crystallography
（研究膜蛋白和脂质平移扩散以及溶质通过质膜和在细胞内转运的一种技术）
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The asymmetric distribution of membrane lipid in plasma membrane
SM:鞘磷脂; PC:磷脂酰胆碱; PS:磷脂酰丝氨酸; PE:磷脂酰乙醇胺; PI: 磷脂酰肌醇; CL: 胆固醇 12
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Studying the structure and properties of membrane proteins
• Peripheral membrane proteins: high- concentration salts solutions • Integral membrane proteins: detergents
Protein
5Байду номын сангаас
1、Cell membrane


Models of membrane structure
Fluid-mosaic model: a membrane consists of a mosaic of proteins in a fluid lipid bilayer
—The “central dogma” of membrane biology
GPI: glycosylphosphatidylinositol (糖基化磷脂酰肌醇 ) 13

The mobility of membrane proteins
• •
Rotational diffusion Lateral diffusion
— Lowering the transition temperature of the lipid bilayer ?