Li T, Johnson J E, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110.
Biophys J. 1993 Nov;65(5):1963-72. doi: 10.1016/S0006-3495(93)81272-4.
We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.
我们描述了一种研究生物组装体中交换动力学的新方法。该方法利用了与透析流通池耦合的拉曼多通道分析仪。我们采用这种方法来确定菜豆斑驳病毒（BPMV）的包装RNA基因组和蛋白质亚基中不稳定氢的交换率。在与人类微小核糖核酸病毒相似的BPMV组装体中，X射线结构表明，尽管有序片段中的核苷酸碱基尚不清楚，但约20%的单链RNA链在二十面体衣壳的三重顶点处是有序的（Chen等人，1989年）。在这里，我们将天然病毒的交换图谱与空衣壳、模型核酸和水性溶剂的交换图谱进行比较，以揭示BPMV RNA和蛋白质的以下交换特征：（i）在包装的RNA中观察到碱基特异性的交换延迟。（ii）尿嘧啶残基的延迟最大，其一级交换速率常数（kU = 0.18 +/- 0.02 min-1）比H2O溶剂或RNA的腺嘌呤或胞嘧啶基团的速率常数低40%（ksolv ≈ kA ≈ kC = 0.30 +/- 0.02 min-1）。（iii）在包装RNA的鸟嘌呤残基中也观察到交换延迟。（iv）在包装RNA或大量溶剂完全交换的时间内（t ≈ 10分钟），衣壳亚基的酰胺NH基团没有明显的交换。因此，目前的结果确定了蛋白质亚基（酰胺NH）和RNA核苷酸（氨基NH2和亚氨基NH）中对溶剂催化的氢交换具有抗性的位点。我们认为RNA核苷酸不稳定位点的交换延迟是病毒粒子内RNA染色体组织的结果。我们的发现支持了一个关于BPMV的模型，其中衣壳亚基的表面和埋藏结构域广泛且刚性地通过氢键结合，并且包装RNA的尿嘧啶和鸟嘌呤外环供体基团是衣壳三重顶点处亚基相互作用的主要靶点。
