College of Physics, Jilin University, Changchun, Jilin 130012, People's Republic of China.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People's Republic of China.
J Chem Phys. 2018 Mar 28;148(12):123331. doi: 10.1063/1.5004205.
A discrepancy has emerged in recent years between single-molecule Förster resonance energy transfer (smFRET) measurements and small angle X-ray scattering (SAXS) or small angle neutron scattering experiments in the study of unfolded or intrinsically disordered proteins in denaturing solutions. Despite significant advances that have been made in identifying various factors which may have contributed to the manifestation of the so-called smFRET-SAXS discrepancy, no consensus has been reached so far on its original source or eventual resolution. In this study, we investigate this problem from the perspective of the solvent effect on FRET spectroscopic ruler (SEFSR), a generic term we use to describe various solvent-dependent factors affecting the accuracy of the FRET experimental method that is known as a "spectroscopic ruler." Some factors belonging to SEFSR, such as direct dye-solvent interaction and labeling configuration, seem to have not received due attention regarding their significance in contributing to the discrepancy. We identify SEFSR by measuring a rigid segment of a double-stranded DNA in various solutions using the smFRET method and evaluate its relative importance in smFRET experiments by measuring segments of a single-stranded DNA and polyethylene glycol (PEG) in solutions. We find that SEFSR can produce non-negligible FRET-inferred interdye distance changes in various solutions, with an intensity following the Hofmeister series in ionic solutions and dependent on labeling configurations. SEFSR is found to be significant in GuHCl and urea solutions, which can fully cover the apparent expansion signal of dye-labeled PEG. Our findings suggest that SEFSR may have played an important role in contributing to the smFRET-SAXS discrepancy.
近年来，在研究变性溶液中无规则或固有无序蛋白质时，单分子Förster 共振能量转移（smFRET）测量与小角 X 射线散射（SAXS）或小角中子散射实验之间出现了差异。尽管在确定可能导致所谓的 smFRET-SAXS 差异的各种因素方面取得了重大进展，但迄今为止，对于其原始来源或最终解决方案仍未达成共识。在这项研究中，我们从溶剂对 FRET 光谱标尺（SEFSR）的影响的角度来研究这个问题，我们使用这个通用术语来描述影响 FRET 实验方法准确性的各种与溶剂相关的因素，该方法被称为“光谱标尺”。一些属于 SEFSR 的因素，如直接染料-溶剂相互作用和标记配置，似乎没有得到应有的重视，因为它们对差异的贡献具有重要意义。我们通过使用 smFRET 方法在各种溶液中测量双链 DNA 的刚性片段来识别 SEFSR，并通过在溶液中测量单链 DNA 和聚乙二醇（PEG）的片段来评估其在 smFRET 实验中的相对重要性。我们发现 SEFSR 可以在各种溶液中产生不可忽略的 FRET 推断出的染料间距离变化，其强度遵循离子溶液中的 Hofmeister 序列，并取决于标记配置。SEFSR 在 GuHCl 和尿素溶液中很明显，它们可以完全覆盖染料标记的 PEG 的表观膨胀信号。我们的研究结果表明，SEFSR 可能在导致 smFRET-SAXS 差异方面发挥了重要作用。
