COOPERATIVE AND NONCOOPERATIVE BINDING OF PROTEIN LIGANDS TO NUCLEIC-ACID LATTICES - EXPERIMENTAL APPROACHES TO THE DETERMINATION OF THERMODYNAMIC PARAMETERS
COOPERATIVE AND NONCOOPERATIVE BINDING OF PROTEIN LIGANDS TO NUCLEIC-ACID LATTICES - EXPERIMENTAL APPROACHES TO THE DETERMINATION OF THERMODYNAMIC PARAMETERS
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DOI：
10.1021/bi00354a006
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发表时间：
1986-03-25
期刊：
影响因子：
2.9
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
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作者：
KOWALCZYKOWSKI, SC;PAUL, LS;VONHIPPEL, PH
许多生物学上重要的蛋白质在发挥其生理功能时非特异性地并且通常协同地结合到单链或双链核酸晶格。这种结合通常可以用热力学术语通过三个参数来描述：n，结合位点大小; K，固有结合常数; ω，绑定协同性参数。这些参数的实验测定往往似乎是直截了当的，但可能充满了概念和方法上的困难，可能不是很明显。在本文中，我们描述和分析了一些方法，可用于测量这些蛋白质-核酸相互作用参数，并说明这些方法与T4编码的基因32（单链DNA结合）蛋白质的各种核酸晶格的结合实验。我们考虑以下程序：（i）滴定固定量的晶格（核酸）与添加的配体（蛋白质）;（ii）用添加的晶格滴定固定量的配体;（iii）在非常低的晶格饱和水平下测定配体结合亲和力;（iv）分析配体簇尺寸分布在晶格上;（v）配体与有限长晶格结合的分析。每种方法的适用性和局限性进行了考虑和讨论，并明确指出潜在的陷阱。
Many biologically important proteins bind nonspecifically, and often cooperatively, to single- or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; .omega., the binding cooperativity parameter. The experimental determination of these parameters often appears to be straightforward but can be fraught with conceptual and methodological difficulties that may not be readily apparent. In this paper we describe and analyze a number of approaches that can be used to measure these protein-nucleic acid interaction parameters and illustrate these methods with experiments on the binding of T4-coded gene 32 (single-stranded DNA binding) protein to various nucleic acid lattices. We consider the following procedures: (i) the titration of a fixed amount of lattice (nucleic acid) with added ligand (protein); (ii) the titration of a fixed amount of ligand with added lattice; (iii) the determination of ligand binding affinities at very low levels of lattice saturation; (iv) the analysis of ligand cluster size distribution on the lattice; (v) the analysis of ligand binding to lattices of finite length. The applicability and limitations of each approach are considered and discussed, and potential pitfalls are explicitly pointed out.