A new tool for measuring surface-biomolecule interactions

描述（由申请人提供）：为什么生物分子附着在某些表面（如细胞膜）时保持活性，而附着在其他表面（如许多人工表面）时几乎总是展开和失活？我们怎样才能重现前一种效果，制造出附着在其上的生物分子同样保持其功能的人造表面？迄今为止，我们解决这些重要问题的能力一直受到严重缺乏定量实验方法来测量生物分子表面相互作用热力学的阻碍。也就是说，尽管大量定性文献描述了吸附如何改变生物分子结构，以及大量寻找抗吸附表面的实证研究，但事实证明，对这种情况发生的方式和原因的定量、实验可测试的见解难以捉摸。因此，我们提出开发和验证一种新的实验（电化学）方法来测量生物分子的折叠自由能，特别是附着在明确定义的宏观表面上。然后与体溶液中的折叠自由能进行比较，从而了解生物聚合物表面相互作用的热力学和机制。迄今为止，我们已经采用这种方法来表征表面受限DNA茎环的易于建模的折叠，这些研究首次通过实验定义了特定生物分子与一系列定义良好的宏观表面相互作用的程度和机制。在这里，我们提出了一个为期两年的研究计划，旨在适应这种定量实验工具，以研究蛋白质表面相互作用。

DESCRIPTION (provided by applicant): 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.