lecture_8_Binding and kinetics_结合与动力学

合集下载

第2章药物代谢动力学

药量---时间关系
血药浓度 A（给药量）可代替C
时
n=1：一级动力学
间
n=0：零级动力学
正值：表示吸收动力学负值：表示消除动力学
一级消除动力学（first-order elimination kinetics
----体内药量以恒定的百分率进行消除(恒比消除（) 掌握）
一级消除动力学特点
----线性动力学(掌握)
pH=7
pH=4
总量 A + H+HA
100001 105
1
HAH+ + A 总量
1
102 101
10pH-pKa =
[ A ] [HA]
= 107-2 = 105
10pH-pKa =
[ A ] [HA]
= 104-2 = 102
问题
某人过量服用苯巴比妥（酸性药）中毒，有何办法加速脑内药物排至外周,并从尿内排出？
F、Vd、 T1/2、 CL
(掌握) 简单扩散
（掌握）
首关消除药酶诱导/抑制尿液PH对药物排泄影响
（掌握）
一级消除动力学零级消除动力学
Css 、F、Vd、 T1/2、 CL
被动转运药物跨膜转运方式
滤过水溶性扩散
简单扩散脂溶性扩散
载
易化扩散
体扩
主动转运
膜
散
动
转
运
1.滤过（Filtration) --水溶性扩散(了解）
3．易化扩散 (Facilitated diffusion; Carrier-mediated diffusion) （了解
）
▲有载体协助的顺差转运，有饱和、竟争现象。

MIT和UM核工程相关课程目录

本科： SH2701: Thermal-Hydraulic in Nuclear Energy Engineering, 6 ECTS credits SH2702: Nuclear Reactor Technology, 8 ECTS credits SH2703: Reactor Dynamics and Stability, 6 ECTS credits Project Course in Nuclear Energy Technology, as a part of course 4A1609 Undergraduate courses in previous years 研究生 Advanced Course in Thermal Hyቤተ መጻሕፍቲ ባይዱraulics, 9 ECTS credits
Nuclear Power Reactors（核电站反应堆） Thermal-hydraulics for Nuclear Systems（热工水力） Reactor Safety Analysis（反应堆安全分析） Introduction to Plasmas（等离子体导论） Fusion Reactor Technology（聚变反应堆技术） Engineering Principles of Radiation Imaging（辐射成像工程原理） Radiological Health Engineering Fundamentals（放射卫生工程基础） Special Topics in Nuclear Engineering and Radiological Sciences（核工程与放射科学专题） Research in Nuclear Engineering and Radiological Sciences（核工程与放射科学研究） 500level Quantum Mechanics in Neutron-Nuclear Reactions（中子核反应中的量子力学） Interaction of Radiation and Matter（射线与材料相互做用） Nuclear Measurements Laboratory（核测量实验室） Advanced Radiation Measurements and Imaging（先进辐射测量和成像） Radiation Materials Science I/II/III（辐射材料科学） Nuclear Fuels（核燃料） Nuclear Waste Management（核废料管理） Detection Techniques of Nuclear Non-proliferation（防扩散探测技术） Nuclear Reactor Theory II（核反应堆理论） Monte Carlo Methods（蒙特卡罗方法） Thermal Fluids for Nuclear Reactor Safety Analysis（反应堆安全中的热工流体） Nuclear Reactor Kinetics（反应堆动力学） Radiation Shielding Design（辐射屏蔽设计） Nuclear Core Design and Analysis I/II（堆芯设计分析） Intermediate Plasma Physics I/II（中间等离子体物理） Plasma Engineering（等离子体工程） Plasma Generation and Diagnostics Laboratory（等离子体产生和诊断实验室） Charged Particle Accelerators and Beams（带点粒子加速器和束流） Plasma Spectroscopy（等离子体光谱学） Physical Processes in Plasmas（等离子体中的物理过程）

毒代动力学

药物经生物转化后，其结局如下：
①灭活、毒性降低极性增加 ③产生毒性代谢物
21
3. 生物转化酶的特点及种类：

专一性酶
如ChE, MAO等
非专一性酶（细胞色素 P450药物代谢酶系，CYP450）生物转化主要在肝脏进行，因促进体内药物生物转化的酶主要是肝脏微粒体氧化酶系统（又称肝药酶）也称为细胞色素P-450氧化酶。其特点是:
32
第二节毒代动力学 Toxicokinetics
在毒理学研究中根据产生毒性作用的剂量，定性和定量地研究实验动物体内药物的吸收、分布、代谢和排泄随
时间的动态变化规律。毒代动力学研究所用的剂量远远高
于药效剂量和临床拟用剂量，并且为多次重复用药，其给
药情况和毒理学研究的实际情况相同或相似，所获结果对
34
（一）经典动力学模型 Classical Ttoxicokinetics 房室概念和房室模型：
动力学的房室(compartment)概念是抽象的数学概念，
其划分取决于毒物在体内的转运及/或转化速率。一房室模型 (one compartment model) 二房室模型 (two compartment model) 中央室 (central compartment)
30
肠肝循环（enterohepatic circulation)
指自胆汁排进十二指肠的结合型药物在肠中经水解后被再吸收的过程。
Liver
Drug
31
经肺排泄
挥发性高的有机溶剂如乙醇等
经唾液、汗腺排泄
铅、砷等重金属和某些生物碱等
经乳汁、头发排泄
许多金属、毒品、有机氯农药等可从乳汁排泄而影响婴儿。某些重金属可排泄到头发中。

基于增强采样分子动力学模拟的蛋白质和小分子相互作用热力学和动力学研究

基于增强采样分子动力学模拟的蛋白质和小分子相互作用热力学和动力学研究摘要蛋白质和小分子相互作用的热力学（结合自由能ΔG bind和平衡解离常数K D）是表征一个药物小分子与其靶蛋白结合稳定性的重要依据，也是评价一个药物小分子与其靶蛋白亲和力大小的重要指标。

而近些年来逐渐受到重视的蛋白质和小分子之间的结合动力学（解离速率常数k off和滞留时间）与药物小分子的药效和毒性等药代动力学性质密切相关，所以在以靶蛋白和药物小分子的热力学性质为依据进行药物设计时应同时考虑它们的结合动力学性质。

基于蛋白质和小分子热力学和动力学的计算方法和预测热力学和动力学的重要性，本论文的研究内容主要有以下五个部分。

本论文第一章详述了蛋白质和小分子相互作用的重要性，从蛋白质和小分子相互作用理论模型开始，介绍了二者相互作用的物理化学基础以及二者结合的热力学和动力学性质。

接着总结了研究蛋白质和小分子相互作用的热力学和动力学的计算方法。

对于热力学性质来说，主要有基于分子对接的打分函数和基于分子动力学模拟的自由能计算方法，如我们熟知的MM/PB(GB)和自由能微扰计算方法。

而针对动力学性质的计算，目前比较成熟的有拉伸分子动力学模拟、自适应偏置力模拟以及meta动力学模拟等增强采样方法。

第二章通过常规分子动力学模拟和拉伸动力学模拟研究了B-RAF激酶的两个高效抑制剂PLX4720和TAK-632解离机制的差异以及解离机制与滞留时间的关系。

从两个抑制剂与B-RAF激酶复合物的晶体结构出发，我们首先对常规分子动力学模拟的平衡轨迹做了能量分解，发现B-RAF激酶结合两个抑制剂的关键氨基酸残基的能量贡献有明显的差异，尤其在变构结合位点处。

这说明变构位点处的疏水作用对于提高B-RAF激酶抑制剂的药效以及延长滞留时间有很重要的作用。

之后我们用随机加速分子动力学模拟对多条平衡轨迹选择不同的参数进行了统计，结果表明抑制剂PLX4720是从ATP通道解离，而抑制剂TAK-632则有1/3的几率从变构通道解离。

药物动力学

Pharmacokinetics
药物代谢动力学
2014.08
药物动力学基本概念
Pharmacokinetics Absorption进入体循环的过程 Distribution向组织转运的过程 Metabolism结构改变 Excretion 排泄
用动力学原理与数学处理方法定量描述药物在体内动态变化规律。新药开发分布生物利用度缓控释制剂临床合理用药给药方案个体化给药
白蛋白albumin (acidic drugs) 糖蛋白glycoproteins (basic drugs) 纤维蛋白元fibrinogen, 脂蛋白 lipoproteins (both, minor)
Compartment Models
example:
A D
Peripheral (fat)
Rate processes (mainly elimination)
zero-order
constant amount of drug eliminated per unit of time elimination rate constant has units of weight/time: K= 50 mg/h, for example
typical drug: ethanol 乙醇
Types of Kinetics Commonly Seen
Zero Order Kinetics

First Order Kinetics

Rate = k C = Co - kt C vs. t graph is LINEAR
Rate = k C C = Co e-kt C vs. t graph is NOT linear, decaying exponential. Log C vs. t graph is linear.

细胞因子接收器和群体感应系统的动态和稳态行为模拟说明书

Supplementary Discussion, Data or MethodsA model was constructed to simulate the dynamic and steady state behavior of the cytokinin receiver and quorum sensing (QS) systems. In the first configuration, the model was used to simulate the receiver’s main regulatory mechanisms including ligand binding and dissociation from the AtCRE1 receptor, auto-phosphorylation of ligand-bound AtCRE1, AtCRE1-YPD1-SKN7 two-component phospho-transfer reactions, SKN7 transcriptional activation, degradation of IP signaling molecule, and cell growth. The second configuration was used to simulate the full QS system by adding the synthesis of IP and continuous culture dilution (only for steady state behavior).Both systems are described by twelve biochemical species and cell population density N (Table 1). Tables 2 and 3 show the systems’ biochemical reactions and the corresponding ordinary differential equations. We assumed the following: (1) increases in cell density follow logistic kinetics 1 with a rate constant k n and saturate with a maximum volume density N max (Table 3, DE1); (2) IP binds AtCRE1 with a cooperativity of one (Table 3, R6 & R8) based on structure predictions showing a one-to-one interaction between the receptor ligand-binding domain and cytokinin 2. Overall system behavior did not change significantly when IP/AtCRE1 binding cooperativity was increased two-fold due to existing cooperativity in SKN7 transcriptional activation as described below. (3) SKN7 forms a protein dimer (SKN7D) that binds two 13bp repeat SSRE sequences (R19 & R20). Activation of SSRE promoter requires SKN7D phosphorylation at two sites (SKN7D PP ) which follows a two-step, distributive mechanism using phosphorelay by YPD1 (R25 & R27). Mono-phosphorylated SKN7D P does not activate transcription; (4) AtIPT4 catalyzes the first and rate-limiting step of IP biosynthesis 3; In the QS configuration, production of IP is therefore directly dependent on the concentration of SKN7D PP (R32). Overall IP production in the culture is also proportional to N (DE2).Table 4 lists the kinetic constants used in the simulations. Most of the values are based on previously published kinetic rates 4-9 with the assumption that 1 nM corresponds to 40 molecules per cell 6. A few parameters for which no published data was available were determined by manuallyfitting to our experimental results and by normalizing the observed fluorescence intensities to GFP concentrations (1 fluorescence a.u. equals to 1 nM). The dynamic behavior of the systems was simulated by using MATLAB’s stiff differential equation solver ode15s.The simulated dynamic and steady state cytokinin receiver behavior is shown in Figure 1. The IP synthesis rate was set to zero and cells were grown without dilution for 6 hours from a density of 2x106 cells/ml to a density of 8x106 cells/ml. Figure 1a shows the time-dependent response of receiver cells grown in media supplemented with 10 M IP. Figure 1b shows the steady-state GFP dosage response to 300 different IP concentrations.Figure 2 shows the simulated dynamic and steady state QS behavior (with IP production). For the dynamic experiment (Fig. 2a ), the SSRE and TR-SSRE QS strains were initially set to low cell densities and to contain high GFP levels and phosphorylated signaling proteins. Figure 2b depicts s teady state GFP concentrations as a function of cell density. The steady state response curves were obtained by simulating 300 different constant cell densities where cells were growing and the media was constantly diluted, with each simulation lasting 36 hours. In order to model constitutive IP expression without positive feedback (GAL1 QS strain), basal IP synthesis rate k bip was increased to 40 and SKN7D PP dependent IP synthesis rate k sip was set to zero. Both the steady state and dynamic simulation results correlate well with our experiments (Figs. 4b and 4c of the main text), demonstrating the important role of positive feedback regulation of AtIPT4 for the switch-like QS response.Reference1. C. Belta, J.S., T. Dang, V. Kumar, G. J. Pappas, H. Rubin, P. V. Dunlap, Stability andreachability analysis of a hybrid model of luminescence in the marine bacterium Vibrio fischeri. in 40th IEEE CDC, (2001).2. Pas, J., von Grotthuss, M., Wyrwicz, L.S., Rychlewski, L. & Barciszewski, J. Structureprediction, evolution and ligand interaction of CHASE domain. FEBS Lett576, 287-290 (2004).3. Kakimoto, T. Biosynthesis of cytokinins. J Plant Res116, 233-239 (2003).4. Mateus, C. & Avery, S.V. Destabilized green fluorescent protein for monitoring dynamicchanges in yeast gene expression with flow cytometry. Yeast16, 1313-1323 (2000).5. Yamada, H. et al. The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor thattransduces cytokinin signals across the membrane. Plant Cell Physiol42, 1017-1023 (2001).6. Klipp, E., Nordlander, B., Kruger, R., Gennemark, P. & Hohmann, S. Integrative model ofthe response of yeast to osmotic shock. Nat Biotechnol23, 975-982 (2005).7. Janiak-Spens, F., Cook, P.F. & West, A.H. Kinetic analysis of YPD1-dependentphosphotransfer reactions in the yeast osmoregulatory phosphorelay system. Biochemistry44, 377-386 (2005).8. Janiak-Spens, F., Sparling, D.P. & West, A.H. Novel role for an HPt domain in stabilizing thephosphorylated state of a response regulator domain. J Bacteriol182, 6673-6678 (2000).9. Janiak-Spens, F. & West, A.H. Functional roles of conserved amino acid residuessurrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1. Mol Microbiol37, 136-144 (2000).10. Mumberg, D., Muller, R. & Funk, M. Yeast vectors for the controlled expression ofheterologous proteins in different genetic backgrounds. Gene156, 119-122 (1995).11. Mumberg, D., Muller, R. & Funk, M. Regulatable promoters of Saccharomyces cerevisiae:comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res22, 5767-5768 (1994).12. Melcher, K., Sharma, B., Ding, W.V. & Nolden, M. Zero background yeast reporterplasmids. Gene247, 53-61 (2000).13. Gueldener, U., Heinisch, J., Koehler, G.J., Voss, D. & Hegemann, J.H. A second set of loxPmarker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res30, e23 (2002).14. Inoue, T. et al. Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature409,1060-1063 (2001).15. Maeda, T., Wurgler-Murphy, S.M. & Saito, H. A two-component system that regulates anosmosensing MAP kinase cascade in yeast. Nature369, 242-245 (1994).16. Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed forefficient manipulation of DNA in Saccharomyces cerevisiae. Genetics122, 19-27 (1989).。

生物化学：Chapter 1-2 Foundations_of_Biochemistry

Sept. 11, 2012
Chapters 1-2
The realm of Biochemistry
Professor Zengyi Chang
(昌增益教授）
changzy@ Room 204, New Life Science Building
Tel. 6275-8822
two books into Chinese!
The eight classic papers (to be read by all students)
Topic 1 the prebiotic Origin of Life
Stanley L. Miller.(1953) “A Production of Amino Acids Under Possible Primitive Earth Conditions”, Science, 117:528-9.
Books on the history of Biochemistry:
1. 昌增益（译者）《蛋白质、酶和基因：化学与生物学的交互作用》，清华大学出版社，2005年1月。
Fruton, J. S. (1999). Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology. New Heaven and London: Yale University Press.
What is life?
Life: A process or condition but a definition not yet achieved.
Possessing the following basic features:
– Homeostasis;

药代动力学总论

Vd＝D／C
设想药物均匀分布于各种组织与体液，且浓度与血液中相同，在这种假设条件下药物分布所需容积。数学概念，并不代表具体的生理空间。给药剂量或体内药物总量与血药物浓度的比例常数
体液总量、组成和药物Vd的关系
血浆 3 L
细胞间液 12 L
细胞内液 27 L 总体液：42 L
Acidic drugs
Non-CYP enzymes
CYP2A6 CYP2B6
CYP2C8
CYP2C9
CYP3A4/5/7
CYP2C19
CYP 2D6 CYP2E1
药物代谢酶的活性可被诱导或抑制
氯苯唑胺(骨松药)浓度（µg/g组织）
药酶诱导 (Induction)：苯巴比妥、利福平，环境
污染物等
无诱导苯巴比妥诱导
• 区分疾病部位给药
– 抗生素、消炎：上呼吸道感染、牙疼、脑膜炎、一般伤口、胃肠道疾病
• 肝肾毒性评估
– 代谢、排泄的主要途径
• 靶向制剂
– 增强药物在靶部位的药效、减小药物毒性
3. 代谢（生物转化)
Metabolism, Biotransformation
代谢部位：主要在肝脏，其它如胃肠、肺、皮肤、肾
药物在体内积蓄和从体内消除时程
87.5% 94% 97%
第四节药物消除动力学 Elimination Kinetics
第二章
体内药物浓度因不断消除而随时间不断变化
k：消除速率常数
(Rate constant for elimination)
dC/dt = - kCn
一级消除动力学 (Firstorder elimination kinetics )
主要内容

化学反应动力学(全套课件582P)

r 1 d[A] 1 d[B] 1 d[C] 1 d[D] a dt b dt c dt d dt
或 r 1 d[Ri ]
i dt
对于气相反应，也可用压力表示反应速率：
rP
1 a
dPA dt
1 b
dPB dt
1 c
dPC dt
1 d
dPD dt
或：
rP
1
i
dPRi dt
对于理想气体： Pi ci RT
化学反应动力学
课程属性：学科基础课学时/学分：60/3
教材:
《 Chemical Kinetics and Dynamics 》 J. I. Steinfeld, et al, 1999 ( Prentice Hall )
参考书：
1《化学反应动力学原理》（上、下册）赵学庄编（高等教育出版社）
k = 2×104
k = 1×10-2
§1-2 反应速率的定义
( Definition of the Rate of a Chemical Reaction ) 若一个反应的化学计量式如下：
(1) a A + b B c C + d D 或写为： (2) 0 = iRi
式(2) 中,
Ri：反应物和产物。 i：化学计量系数, 它对于反应物为负,
1 给定乙醛的初始浓度, 测定不同反应时间的反应速率及乙醛浓度，从而确定反应的反应级数。
则反应速率与[CH3CHO]的平方成正比，即称其时间级数为二级的。
2 以乙醛的不同初始浓度进行实验, 测定不同初始浓度下的反应速率，从而确定反应级数。
则反应速率与乙醛的初始浓度的一次方成正比，即称其浓度级数为一级的。

药理学第二章药物代谢动力学

• ATP：Na+：K+＝1：3：2
当细胞内[Na+]升高或细胞外[K+]升高时,钠泵被激活。分解ATP供能，将Na+泵出细胞,同时将K+泵入细胞.
（2）继发性主动转运（secondary active transport）：又称二次性主动转运。即不直接利用分解ATP产生的能量，而是与原发性主动转运中的转运离子相耦合，间接利用细胞内代谢产生的能量来进行转运。这种转运使物质跨膜转运的最普遍方式。
三、代谢（生物转化, metabolism, biotransformation）
指药物在体内发生的化学结构改变。转化后的大多数药物药物活性降低或失去药理活性，极性增加，易于排泄。
部位：主要在肝脏，
其它如胃肠、肺、皮肤、肾。
代谢主要由细胞色素P450单氧化酶系（简称“肝药酶”）催化。
药物氧化代谢 (Oxidation) 细胞色素P450单氧化酶系（CYP）
需特异性载体。
如体内葡萄糖和一些离子（Na+、K+、 Ca2+等）的吸收。
顺浓度梯度，不耗能，不能逆浓度梯度转运。
2、主动转运（active transport）：
药物从低浓度一侧跨膜向高浓度一侧的转运，又称逆流转运、上山运动。
主动转运的特点：（1）药物逆浓度差转运（2）耗能（3）需要载体（4）有饱和现象及竞争性抑制
药物跨膜转运速度符合Fick定律
通透量（分子数/min） =（C1-C2）× 膜面积×通透系数
膜厚度
C1-C2 为药物浓度差，通透系数即药物分子的脂溶度
（二）药物转运体
药物转运体（transporter）是跨膜转运蛋白，是药物载体的一种。转运体可分为：

内向整流钾通道门控动力学过程的研究

河北工业大学硕士学位论文内向整流钾通道门控动力学过程的研究摘要Kir2.1通道是一种广泛分布于心脏、神经系统、平滑肌等许多组织中的钾离子通道。

它具有很强的内向整流特性，在诸多生理过程中都起到关键作用，例如：心律的控制，调节神经元兴奋性和激素分泌，参与了脑细胞中胞外钾离子的转运及肾脏细胞中钾离子的分泌等等。

膜磷脂PIP2是Kir2.1通道功能的重要调节因子，通道开放与否依赖于PIP2与通道是否结合。

在Kir2.1通道中，有三个门，第三个门位于通道C-末端（如图4.1.1），Kir2.1通道与PIP2的结合位点也位于C-末端。

目前，关于第三个门门控的调节机制以及PIP2与通道结合以后相互作用力产生传递的分子基础尚不清楚，有待进一步研究。

本文以Kir2.1通道三维结构为基础，采用分子生物学实验、双电极电压钳实验以及膜片钳实验等手段，结合分子对接、分子动力学模拟及施力分析等理论研究方法，研究了PIP2门控Kir2.1通道的微观机制。

我们通过实验研究发现：突变体通道E303A、A304G、V223L、V223M可以表达为功能性通道，E303D、E303Q及E303V不能表达为功能性通道。

其中突变体通道的全细胞电流峰值明显低于野生型通道,它们对PIP2的依赖性也有所改变，然而，对内源性及外源性的PIP2依赖性有着相同的模式。

为解释我们所观察到的实验现象，我们进行了理论研究，其初步研究结果表明PIP2门控Kir2.1通道过程依赖于通道与PIP2分子之间的相互作用，其中氢键弱相互作用起到了重要作用。

由于点突变诱发的全局构象变化，对于PIP2门控Kir2.1通道过程也起到了重要作用。

研究发现，多种离子通道的功能都受到膜磷脂PIP2的调控作用，然而对于PIP2门控离子通道的理论研究刚刚起步，本文的研究仅仅涉及到一种钾离子通道，在我们后续的工作中会考虑更多离子通道与PIP2相互作用的研究。

关键词：Kir2.1通道，PIP2，门控，分子动力学，氢键i内向整流钾通道门控动力学过程的研究ii Study on the Gating Kinetics of Inward Rectifying K+ ChannelsABSTRACTKir2.1, Inward rectifying K+ channel, is widely distributed in many tissues, such as cardiacmuscle, nervous system etc. Kir2.1 channel shows "strong" inward rectification and plays a vital role in many diverse physiological processes, including control of the heart rate, setting the activation threshold of neuronal excitability, hormonal secretion, extracellular K+ buffering in the brain, and K+ secretion in the kidney. PIP2 (phosphatidylinositol 4,5-bisphosphate) is one of the most important modulators of the Kir2.1 channel. The transition between open and close of Kir2.1 channel depends on whether PIP2 combines with the channel or not. There are three gates in Kir2.1 channel. The third gate locates at the C-terminal domain of Kir2.1, at which the binding sites of PIP2 locate (Figure 4.4.1). The questions now are how does the third gating mechanism work and how the interaction force between PIP2 and the channel produces and transmits.Based on the three-dimensional structure of Kir2.1 channel, we used molecular biology experiment, double electrode voltages clamp experiment and patch clamp experiment to study the kinetics of PIP2 modulating wild type (WT) Kir2.1 and its mutants. We also used molecular docking, molecular dynamics simulation and steered molecular dynamics (SMD) method to study essential motion mode during free or constrained dynamics and structural response during forced pulling of Kir2.1 cytoplasmic domains. Our data show that the whole cell peak currents of Kir2.1 mutants are significantly lower than those of the wild type channel. Comparing the dose-dependent relation between WT and mutant Kir2.1 chanenls, the affinities of PIP2 are different. However, the kinietics of endogenous and exogenous PIP2 modulating WT and mutant Kir2.1 channels have the same pattern. To explain what we observed experimental phenomena, we do theoretical research. Preliminary research results show that the PIP2 gating Kir2.1 channel process relies on the PIP2 binding with channel and the interaction which is mainly based on the hydrogen bonds. The global conformational changes which are induced by point mutations, also河北工业大学硕士学位论文play important roles in the process of PIP2 modulating Kir2.1 channels.As we know that the functions of many ion channels are modulated by PIP2. However, in our study, only the Kir2.1 channel and its mutants are researched which is not enough to elucidate the mechanism how PIP2 modulates the ion channels. In our follow-up work, we will consider more ion channels which can be modulated by PIP2.KEY WORDS: Kir2.1 channel, PIP2, gating, molecular dynamics, hydrogen bondiii原创性声明本人郑重声明：所呈交的学位论文，是本人在导师指导下，进行研究工作所取得的成果。

陈述物理化学热力学在药学或生物学领域中的研究文献

陈述物理化学热力学在药学或生物学领域中的研究文献热力学在药学和生物学领域中的应用是广泛的，以下是一些探讨热力学在这些领域中的研究文献的例子：1. "Thermodynamics and Kinetics of Drug Binding to Receptors" (药物结合受体的热力学和动力学)，由Born, Jancso和Bohman 于2009年在Current Medicinal Chemistry杂志上发表。

该研究探讨了药物与受体之间的相互作用，并使用热力学方法研究了药物结合和解离的过程。

2. "Thermodynamic Analysis of Protein Folding" (蛋白质折叠的热力学分析)，由Thirumalai和Woodson于2010年在Annual Review of Biophysics杂志上发表。

该研究利用热力学原理研究了蛋白质折叠的过程，并解释了蛋白质折叠的稳定性和动力学特性。

3. "Thermodynamics of Lipid Membrane Interactions with Drugs" (药物与脂质膜相互作用的热力学)，由Boggs于2009年在Biochimica et Biophysica Acta杂志上发表。

该研究探讨了药物与细胞脂质膜之间的相互作用，并使用热力学方法研究了这些相互作用的热力学特征。

4. "Thermodynamics of Enzyme-Catalyzed Reactions" (酶催化反应的热力学)，由Benkovic和Bunville于2018年在Annual Review of Biochemistry杂志上发表。

该研究利用热力学原理研究了酶催化反应的动力学和热力学特征，并解释了酶催化反应的速率和选择性。

5. "Thermodynamics of Drug-Target Interactions" (药物-靶标相互作用的热力学)，由Mobley和Dill于2009年在Annual Review of Biophysics杂志上发表。

RelaxationKinetics：弛豫动力学

Winter 2006Experiment C4 Chemistry 114HRelaxation KineticsTHEORYWhen a chemical reaction system, in a state of equilibrium, is subjected to a perturbation, it will relax to a new equilibrium position. For kinetic measurements, the perturbation (such as a change in temperature, or pressure, or concentration, etc.) should be accomplished in a time interval which is very small compared to the time scale of the relaxation process. If the new equilibrium position is not far from the initial state, then the kinetics will approximate to first order:dx /dt =!kx (1)where x is the displacement with respect to the final equilibrium, t is the time, and k the rate constant. For example, if the concentration of a reactant (or product) is measured as a function of time, then x = | C(t) - C(∞) | > 0 (2)where C(t) is the concentration at time t and C(∞) is the concentration at equilibrium.The relaxation time is defined asτ = 1/k. (3) Integration of equation (1) gives:ln x = ln x o - t/τ (4) Here x o = | C(0) - C(∞) | > 0(5) A plot of ln x vs t gives: τ = - 1/slope (6)In general, provided the perturbation is sufficiently small and fast , any property, Y, of the system which varies with time may be used. The displacement from equilibrium is then given byx =Y t ()!Y "()>0 (7)Temperature jump and relaxation kinetics were used by Eigen and de Maeyer (1955) to measure the forward rate constant of the reaction [M. Eigen and L. de Maeyer (1955) A. Elektrochem. 59, 986] H 3O ++OH !"H 2O +H 2O k f=1.4x 1011M !1s !1at 25o C Relaxation kinetics following a concentration jump was applied by Swinehart and Castellan (1964) to the slow bichromate-dichromate reaction at 22˚C (τ of the order of 10 s):HCrO 4!"K a H ++CrO 42! (Rx. 1) 2HCrO 4!"k r k fCr 2O 72!+H 2O (Rx. 2) The acid dissociation equilibrium (Rx. 1) is very fast compared to the forward and reverse rates of the dimerization (Rx. 2). [J. H. Swinehart and G. W. Castellen (1964) Inorg. Chem. 3, 278-280; J. H. Swinehart (1967) J. Chem Educ. 44, 524-526]Experiment C4Chemistry 114H -2- Pertinent equations for this experiment are summarized below:K a = f([H +] [CrO 42-] [HCrO 4-]) = 7.4 ∗ 10−7 M (8)Dimerization constant: K d = [Cr 2O 72-] / [HCrO 4-]2 = k f /{k r [H 2O] = 50 M -1 (9)Relaxation time: 1/τ = 4k f [HCrO 4-] + k r [H 2O](10) Calculate the equilibrium [HCrO 4-] as a function of the total chromium concentration, the dimerization and dissociation constants, and the pH (or [H +]) (Hint : write down the conservation of mass for chromium and combine this equation with the two equilibrium constants; you will arrive at a quadratic equation on [HCrO 4-])EXPERIMENTAL PROCEDUREPrepare 50 ml of solution B and the necessary amount of 0.1 M KNO 3Solution A:0.01 M K 2Cr 2O 7 (= 0.02 M in Cr) in 0.1 M KNO 3 Solution B: 0.2 M K 2Cr 2O 7 (= 0.4 M in Cr) in 0.1 M KNO 3Place 50 ml of solution A in a beaker equipped with magnetic stirring and adjust to pH 7 with NaOH. Inject solution B using a syringe while stirring. The pH varies with time as the reaction proceeds.Repeat last step for at least 5 different volumes of solution B between 0 and 1 ml; and repeat each volume 3 timesTo calibrate the pH meter follow the instructions on the computer desktop. Remember to input the calibration constant and the potential measured at pH=7 in the LabView program. The voltmeter should be in the mV scale.RESULTSPlot:ln x vs t, slope = -1/τ Plot:1/τ vs [HCrO 4-], slope = 1/k f , intercept = k r [H 2O] Compute: K d = k f /{k r [H 2O]}You will probably note that the computed K d does not entirely agree with that assumed in Eq.(9) and used to calculate [HCrO 4-]. Does it lie within the estimated error limits? Would it be worthwhile to repeat the calculations with a different K d ?What about the variation of [HCrO 4-] during the run - before equilibrium is reached? Calculate the % change in total Cr concentration for every experiment.What is the advantage of starting the reaction at ph=7?。

中间体的吸附、偶联等反应

中间体的吸附、偶联等反应英文回答：The adsorption and coupling reactions of intermediates play crucial roles in various chemical processes. These reactions are often involved in the formation of new chemical bonds and the transformation of reactants into products. Understanding the mechanisms and kinetics of these reactions is essential for designing efficient catalytic systems and optimizing reaction conditions.Adsorption is the process by which molecules or atoms bind to the surface of a solid or liquid. It can occur through various interactions, such as van der Waals forces, hydrogen bonding, or covalent bonding. The adsorption of intermediates onto a catalyst surface is often the first step in a catalytic reaction. It provides a platform for further reactions to take place and influences the overall reaction rate.Coupling reactions involve the formation of new chemical bonds between two or more reactant molecules. These reactions are commonly used in organic synthesis to construct complex molecules. In many cases, the intermediates formed during the reaction play a crucialrole in the coupling process. They can undergo various transformations, such as oxidative addition, reductive elimination, or nucleophilic attack, to form the desired products.The study of adsorption and coupling reactions of intermediates is often carried out using various experimental techniques, such as surface science methods, spectroscopy, and kinetic measurements. These techniques provide valuable information about the nature of the intermediates, their binding sites on the catalyst surface, and their reactivity. Computational methods, such as density functional theory (DFT) calculations, are also widely used to investigate the reaction mechanisms and energetics of these processes.Understanding the factors that influence the adsorptionand coupling reactions of intermediates is crucial for catalyst design and optimization. The nature of thecatalyst surface, including its composition, morphology, and crystal structure, can significantly affect the adsorption and reactivity of intermediates. The presence of co-adsorbates or additives can also influence these reactions by modifying the electronic properties of the catalyst surface or providing additional reaction pathways.In conclusion, the adsorption and coupling reactions of intermediates are important processes in various chemical reactions. Studying these reactions can provide valuable insights into the mechanisms and kinetics of catalytic processes. By understanding the factors that influence these reactions, researchers can design more efficient catalysts and optimize reaction conditions for various applications.中文回答：中间体的吸附和偶联反应在各种化学过程中起着关键作用。

促红细胞生成素突变体研究

生物制药与研究2019·02189Chenmical Intermediate当代化工研究促红细胞生成素突变体研究＊郭家榕（清华大学附属中学北京 100084）摘要：随着计算机运算能力的飞速发展，用分子动力学模拟等方法进行分子设计及蛋白突变研究取得了长足进步。

本论文分析了促红细胞生成素（EPO）的氨基酸序列及二级结构组成，然后对EPO进行定点突变，提高EPO和EPOR结合表面的静电互补并理论计算结合速率常数，研究发现D43K和I133K双突变可以提高结合速率常数约20倍，大幅提高了EPO-EPOR结合的亲和力。

该论文的研究为研究者运用计算手段设计其他体系突变体蛋白提供了重要的理论实践和方法指导。

关键词：蛋白突变；静电互补；结合动力学中图分类号：R 文献标识码：AStudy on Erythropoietin MutantGuo Jiarong(The Attached High School of Tsinghua University, Beijing, 100084)Abstract ：With the rapid development of computer computing power, great progress has been made in molecular design and protein mutationresearch by the method of molecular dynamics simulation. In this paper, the amino acid sequence and secondary structure of erythropoietin (EPO) were analyzed, and then the EPO was mutated at site to improve the electrostatic complementation of EPO and EPOR binding surface and calculate the binding rate constant theoretically. It was found that D43K and I133K double mutation can increase the binding rate constant by about 20 times, which greatly improved the affinity of EPO-EPOR binding. The research in this paper provides important theoretical practice and methodological guidance for researchers to design mutant proteins of other systems by means of calculation.Key words ：protein mutation ；electrostatic complementation ；binding kinetics1.引言促红细胞生成素（英文名称：Erythropoietin，简称：EPO），是一种人体内源性糖蛋白激素，其可以刺激红细胞的生成，在缺氧条件下，促红细胞生成素生成增加，导致红细胞增生。

变构酶动力学总结

酶的变构调节动力学（总结 2010/8/17 neobe）单底物反应和 Michaelis-Menten kinetics虽然反应机理或许很复杂，但常以来表示单底物反应模型。

如果反应有中间体，由多个基元反应构成，Kcat则是基元反应速率常数的一个函数，如果是单独的一个基元反应，则Kcat就等于这个反应的速率常数。

Kcat又叫作转换数 turnover number, 它表示每秒每催化位点（或者每个酶）转化的最大底物数目。

米氏方程米氏方程是用来描述初始反应速率与底物绑定（离解平衡）以及速率常数关系的一个方程：我很遗憾地说，记得最初接触这个方程立马就熟记的滚瓜烂熟，可是在其后的很长一段时间，关于一些具体问题的理解后来才发现是不深入的甚至是错的。

可是我现在却不想多说，我只想说：看式子！看式子，[S]很大很小，会大约怎么样。

看式子，不论底物浓度如何，只要反应速率始终与酶浓度呈正比。

就是说哪怕底物一丁点，只要增加酶量，速率同样会成倍增加，这说明酶是总体协作的而不是一部分被空余闲置不起作用。

当然米氏方程有诸多假设条件：（1）准稳态（2）总酶量恒定(3)没有中间体，没有底物抑制，或变构调节或协同性。

绑定平衡底物绑定数A primary method for investigating regulatory enzymes is the study of substrate and effector binding to the enzyme. Therefor, the annalysis of equilibrium binding isotherms is now reviewed. 如果蛋白分子P，对配体L有n个绑定位点，则离解常数和绑定平衡如下：酶催化过程中，在一定的底物浓度（或其他结合物浓度）下，酶绑定的平均底物分子数目用r 来表示：这个式子有时又叫作Adair方程。

如果绑定位点互相独立并且相似（没有协同性），则上式中离解常数Ki都可以用一固有的离解常数来表示：带入后整理可得：此式经过变换，1/r 对1/L作图，或者 r/L 对r作图（Scatchard plot）都是线性的。

酶动力学及抑制剂第二讲1-18

二次作图
双倒数作图的斜率对[I]作图为直线，此二次作图的斜率为Km/VmKis，Y截距为 Km/Vm。以二次作图的Y截距除斜率可得 Kis。
双倒数作图的Y截距对[I]作图为直线，此二次作图的斜率为1/VmKii，Y截距为 1/Vm。以二次作图的Y截距除斜率可得 Kii。
小结
1）在可逆抑制剂存在时，固定[I]后，双倒数作图为直线。
5.一般非竞争性抑制(混合型)
公式：
Kis < Kii <∞ , 为竞争性和非竞争性混合型抑制。 Kii < Kis <∞ , 为反竞争性和非竞争性混合型抑制。
公式：
双倒数作图
竞争性和非竞争性混合，直线交于第二象限：
斜率：
Y截距：
双倒数作图
反竞争性和非竞争性混合，直线交于第三象限：
斜率：
Y截距：
1）变化底物：选定底物（考察竞争性的）。按双倒数作图实验要求设计底物浓度。
2）选定3－5个抑制剂浓度，最小可以是0，最大可以在抑制程度50％－70％的浓度，其它均匀分布其间。
3）以抑制剂浓度为固定变化，测定不同底物浓度的反应速度。注意抑制剂是直接加到测活溶液中再加酶启动反应的。
4）操作注意点和双底物动力学实验相同。
导出[ES]/[ET]表达式
得到 v/Vm 公式
以Km 代Ks
比较米氏公式
(2-4)
三、抑制剂和底物的几种竞争关系
1. 概念
抑制剂和底物的竞争关系可提供有关它们在酶上的作用以及酶的活性部位的重要信息。
竞争性抑制示意图
反竞争性抑制示意图
非竞争性抑制示意图
2. 竞争性抑制
双倒数作图
二次作图
斜率对[I]作图为直线，此二次作图的斜率为Km/VmKi，Y截距为Km/Vm。以二次作图的Y截距除斜率可得Ki。

Fortebio_分子相互作用仪讲座PPT

• 更宽的应用范围
• • • • 直接检测粗制的样品，甚至是样品中存在不溶解的成分耐受各种溶液环境，只有结合到传感器表面的分子才会被检测病毒颗粒等大分子样品也能得到动力学结果检测共价键结合的样品
• 更低使用和维护的成本
• 相对其他的非标记检测系统，耗材的成本低 • 系统稳定，未来的使用和维护成本也低
传感器种类12种
Application Quantitation Sensor Type Anti-Human IgG Fc Anti-Murine IgG (Fab’)2 Protein A/G/L Streptavidin (SA) Anti-Penta-HIS (HIS) Regeneration Yes Yes Yes Yes
Y = Y0 + A (1 - e-kobs*t) KD =
Y=Y0+Ae-kd*t kd ka
ka=
kobs – kd
[Conc Ag (M)]
Octet 系统测定动力学的工作流程
Baseline Baseline Loading
Octet Biosensors
Buffer Ligand-Biotin Protein of Interest
Available as option for all systems
96 HA 384 TW 96 well microplate with half-area well size ForteBio 384 tilted-well microplate
1 mM to 10 pM
Octet 384 系统的优点
– SDi 2010 Market Report survey – 成长最快速的非标记技术公司

受体放射分析 (2)

所以容易饱和。但是，任何受体标本除特
异结合外，还会有一部分非特异结合，当
分离特异结合的复合物测量放射性时，这
部分非特异结合的放射性混在一起，测到
的是总放射性（total binding，TB），
必需先减去非特异结合（NSB），才能得
到特异结合（specific binding，SB）的
放射性。
整理课件
整理课件
3、特异性强即该配基与所研究受体有选择性结合，理想的是该配基只与一种受体或受体亚型结合。但没有一种配基是完全选择性的，所以要结合实验研究的目的，选择对某一受体或其中某一亚型亲和力高的放射性配基。 4、稳定性好包括标记的稳定性、贮藏时的稳定性以及温育分析时之稳定性。
从实验目的考虑则主要Байду номын сангаас对具体研究目标来选择。例如有的受体有几种亚型，如果实验的意图是放射性配基要结合所有这几个亚型，即可选择无亚型选择的配基。相反，想研究其中某一个亚型，则应选择针对该亚型的高亲和力配基。
整理课件
第三节受体放射分析的基本方法
一、放射性配基的要求
制备优良的放射性配基是受体结合试验的首要条件。对任何一种受体系统，通常都有好几种标记配基可供选择。选择应从两方面考虑：①配基的特性；②实验目的。
（－）对放射性配基的基本要求
1、高比活度组织细胞内的受体浓度一般都很低，约在 104-105 个／细胞，或 0.013.0pmol/mg蛋白的水平，而且受体对配基的平衡解离常数约在10-9mol/L上下。因此，低比活度的配基难于达到分析灵敏度的要求。
严格构型和构象的配基分子才能选择性地与受体结合。受体的特异性还表现在器官或组织（靶器官）的专一性上，如雌激素对子宫、阴道、乳腺等器官有兴奋作用，这是因为这些器官上雌激素受体的数量明显高于非靶器官。受体的亲和性是指受体和配基的结合能力，受体亲和性高就说明受体和配基结合的牢固，不容易解离。受体亲和性的定量指标就是受体的平衡解离常数KD值。受体的KD值一般在10-8-10-10mol/L之间，KD值在109mol/L以上的受体，一般认为是高亲和性。

等温滴定量热法

▪Lead optimization.先导物优化 ▪Enzyme kinetics.酶动力学 ▪Assessment of the effect of molecular structure changes
on binding mechanisms.分子结构变化对绑定机制评估的影响 ▪Assessment of biological activity.评估生物活性
▪ 可获得生物分子相互作用的完整热力学参数，包括结合常数（Ka）、结合位点数（n）、摩尔结合焓（△H）、摩尔结合熵（△S）、摩尔恒压热容（△Cp），和动力学参数 (如酶促反应的Km和kcat）,用来表征生物分子间的相互作用。
Page ▪ 13
.
13
独特特点：
– 样品用量小，方法灵敏度和精确度高(仪器最小可检测热功率2 nW，最小可检测热效应0.125uJ，生物样品最小用量0.4ug，温度范围2 ℃ - 80 ℃，滴定池体积(1.43 ml)。
Page ▪ 3
灵敏度的提高
.
3
history
Since the beginning of 1990s, the number of published papers related to ‘‘isothermal titration calorimetry ’’ has symptomatically increased due to rapid diffusion of new commercial calorimeters in the scientific community.
结合位点数不同，平衡常数的物料平衡的表达公式是不同的
Page ▪ 11
.
11
Page ▪ 12
.