A new tool for measuring surface-biomolecule interactions

项目总结。为什么生物分子在附着在某些表面(如细胞)时保持活性 膜)，但几乎总是在附着于其他膜(例如，许多人工膜)时展开和失活 表面)？我们如何才能重现以前的效果，以产生附着在其上的人造表面 生物分子同样保留了它们的功能？迄今为止，我们解决这些重要问题的能力一直是 由于严重缺乏测量热力学的定量实验方法 生物分子-表面相互作用。也就是说，尽管有大量定性文献描述了 吸附改变了生物分子结构，大量的实验研究寻找吸附- 抗性表面，定量的，可实验测试的对这种情况发生的方式和原因的洞察力已经被证明 难以捉摸。因此，我们在此建议开发和验证一种新的实验 (电化学)测量特定结合部位的生物分子折叠自由能的方法 定义良好的宏观表面。与本体溶液中折叠自由能的比较，进而揭示 生物聚合物-表面相互作用的热力学--以及由此而来的机制。到目前为止，我们有 用这种方法表征了易于建模的表面受限DNA茎环的折叠 这些研究第一次从实验上定义了一种 特定的生物分子与一系列定义明确的宏观表面相互作用。在这里，我们建议两年 旨在将这一定量实验工具应用于蛋白质表面研究的研究计划 互动。

Project summary. Why do biomolecules retain activity when attached to some surfaces (e.g., the cell membrane) and yet almost invariably unfold and inactivate when attached to others (e.g., many artificial surfaces)? And how can we recreate the former effect to produce artificial surfaces on which attached biomolecules similarly retain their function? To date our ability to address these important questions has been hampered by an acute lack of quantitative experimental methods for measuring the thermodynamics of biomolecule-surface interactions. That is, despite a large body of qualitative literature describing how adsorption alters biomolecular structure, and a large number of empirical studies searching for adsorption- resistant surfaces, quantitative, experimentally testable insights into how and why this occurs have proven elusive. Thus motivated, we propose here the development and validation of a novel experimental (electrochemical) approach to measuring the folding free energy of biomolecules site-specifically attached to well-defined macroscopic surfaces. Comparison with the folding free energy in bulk solution then informs on the thermodynamics -and thus mechanisms- underlying biopolymer-surface interactions. To date we have employed this approach to characterize the easily modeled folding of a surface-confined DNA stem-loop in studies that have, for the first time, defined experimentally the extent to which, and mechanisms by which a specific biomolecule interacts with a range of well-defined, macroscopic surfaces. Here we propose a two-year research program aimed at adapting this quantitative experimental tool to the study of protein-surface interactions.