《蛋白质的结构测定》PPT课件

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1、第一节　蛋白质溶液的热力学第二节　蛋白质折叠动理学第三节　突变、稳定性和折叠热运动与蛋白质构象热力学函数与热力学平衡热容量van’tHoff焓折叠/退折叠转变量热法与折叠过程热力学第十讲蛋白质的物理化学性质分析第一节　蛋白质溶液的热力学第二节　蛋白质折叠动理学第三节　突变、稳定性和折叠一、折叠动理研究技术二、两态动理三、过渡态折叠过程与过渡态对折叠过渡态的性质分析四、折叠中间态熔球态快态与慢态二硫键引起的中间态多结构域蛋白的折叠第十讲蛋白质的物理化学性质分析五、折叠的基本过程接触形成螺旋-链环转变-发卡形成第一节　蛋白质溶液的热力学第二节　蛋白质折叠动理学第三节　突变、稳定性和折叠二、突变与

2、热稳定性疏水突变氢键突变适应极端条件的突变体一、热力学参数在分子水平上的解释静电相互作用范德华相互作用氢键疏水效应二硫键第十讲蛋白质的物理化学性质分析三、突变与折叠过程过渡态的突变分析突变对稳定性和折叠过程的影响NuclearMagneticResonance(NMR)X-rayCrystallographyCryo-ElectronMicroscopy(cryo-EM)第十一讲蛋白质的结构测定NuclearMagneticResonance(NMR)X-rayCrystallographyCryo-ElectronMicroscopy(cryo-EM)Doesnotneedcrystals.

3、Moreinformationaboutdynamics.LimittoMW<40,000.Lessreliable.++--Atomicresolution.+Unlimitedsize.+Time-resolvedstudycanbeperformed.+Techniquesveryrobust.+Needcrystals.-Dealwithverylargecomplex.+Varioussampleformscanbestudied.+Lowersamplepurity.+Doesnotneedcrystals.+-Complicatedexperimentalprocedures.L

4、owerresolution.-第十一讲蛋白质的结构测定X-rayCrystallography-1AnalogiesBetweenLightMicroscopyandX-rayCrystallography.X-rayCrystallography-2WhyX-ray??X-raysareneededtovisualizeatomicfeatures.X-rayswavelengths:CuKa=1.54Å,Synchrotron=0.5~2.5ÅTheoreticalresolutionlimit:Rl/2WhyCrystal??NolenscanfocusX-ray.Crystalsr

5、atherthansinglemoleculesareusedmerelytoboostsignallevel.(Crystalsareregularperiodicarraysofmolecules.)X-rayCrystallography-3X-rayCrystallography-4KeyStepsofMacromolecularStructureDeterminationbyX-rayCrystallographySamplepreparation.Growcrystals.CollectX-raydiffractiondata.Estimatephasesforthediffrac

6、tionpattern.Calculateanelectrondensitymap.Buildanatomicmodel.Refinethemodel.Interpretthemodelaccordingtoavailablebiochemical/geneticdata.Designnewstructural/biochemical/mutagenicexperiments.X-rayCrystallography-5ATypicalSolubilityCurveforaProteinX-rayCrystallography-6TheVaporDiffusionMethodofCrystal

7、lizationX-rayCrystallography-7AStandardApproachofFindingInitialCrystallizationConditionpH+precipitantCrystalsofBio-MacromoleculesProteincrystalstypicallyContain~30%to80%solvent.Proteincrystalsusuallyc