PDF(Pubmed)
Abstract：
Life on the molecular scale is based on a versatile interplay of biomolecules, a feature that is relevant for the formation of macromolecular complexes. Fluorescence-based two-color coincidence detection is widely used to characterize molecular binding and was recently improved by a brightness-gated version which gives more accurate results. We developed and established protocols which make use of coincidence detection to quantify binding fractions between interaction partners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsically related to single-molecule detection, the concentration of diffusing molecules for confocal detection is typically in the low picomolar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliability of our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity at different temperatures, we were able to determine the thermodynamic parameters of the binding interaction. The results show that the ultra-tight binding is dominated by entropic contributions.
摘要：
分子尺度上的生命是基于生物分子的多种相互作用，与大分子复合物形成相关的特征。基于荧光的双色重合检测被广泛用于表征分子结合，最近通过亮度门控版本进行了改进，该版本提供了更准确的结果。我们开发并建立了协议，该协议利用重合检测来量化用不同颜色的荧光染料标记的相互作用伴侣之间的结合分数。由于所应用的技术与单分子检测本质上相关，用于共聚焦检测的扩散分子的浓度通常在低皮摩尔范围内。这使得该方法成为确定双分子结合亲和力的强大工具，就KD值而言，在这个政权。我们通过分析非常强的纳米抗体-EGFP结合证明了我们方法的可靠性。通过测量不同温度下的亲和力，我们能够确定结合相互作用的热力学参数。结果表明，超紧密结合主要由熵贡献。
