sectionb蛋白质结构proteinstructurecentraldogma中心法则.ppt

蛋白质的功能 1 催化功能enzyme 酶 2 信号传递 cell membrane protein 3 转运与贮存 hemoglobin transports oxygen 4 结构与运动 collagen, keratin, tubulin in cytoskeleton, actin and myosin for muscle contraction 5 营养casein (酪蛋白) and ovalbumin(卵清蛋白) 6 免疫 antibodies 7 调节 transcription factors 8 抗癌药物毒蛋白 9 支持与保护作用毛发的角蛋白 + Electrophoresis Protein migrate at different position depending on their net charge Isoelectric focusing A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel. 3. Hydrophobicity(疏水性): hydrophobic interaction chromatography Similar to ion-exchange chromatography except that column material contains aromatic(芳香族的) or aliphatic alkyl(脂肪烷基) groups 4. Affinity chromatography 亲合色谱法 ding d Enzyme-substrate binding Receptor-ligand binding Antibody-antigen binding Substrate analogs: competitive inhibitors 5. Recombinant techniques: Clone the protein encoding gene of interest in an expression vector with a purification tag(纯化标签) added at the 5’- or 3’ end of the gene Protein overexpression in a cell Protein purification with affinity chromatography. Determine the primary structure of a protein: p Amino acid composition: Acid treatment to hydrolyze peptide bonds: 6M HCl, 110°C for 24 hrs. Chromatographic analysis色谱[层]分析 However, you cannot get the sequence! Protein sequence analysis (1) Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV Trypsin T1 HLMGSHLVDALELVMGDR T2 GFEYTPK T3 AWLV V8 V1 HLMGSHLVDALE V2 LVMGDRGFE V3 YTPKAWLV Mass Determination ?Gel filtration chromatography and SDSComparing of the unknown protein with a proper standard Popular SDS: cheap and easy with a 5-10% error SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass. Mass Determination Mass spectrometry质谱分析: Molecules are vaporized and ionized (by Xe/Ar beam), and the degree of de