Probing Macromolecules at the Molecular Level by Pyrene Excimer Fluorescence

荧光共振能量转移（FRET）无疑是最著名的荧光技术探测内部长度尺度（ILSs）和内部动力学（IDy）的溶液中的大分子。这些参数很重要，因为它们解释了大分子的作用。由于大分子具有众多的工业应用，因此对其的研究不仅从基础科学的角度来看，而且对加拿大的经济也具有重要意义。FRET的原理是作为一个光谱尺子。FRET速率常数（kFRET）取决于能量供体和能量受体染料之间的距离。这种关系对于通过测量附着在大分子特定位置的两种染料之间的kFRET来测量刚性大分子的大小具有相当大的价值。不幸的是，对于具有多个距离的标记位点的柔性大分子来说，这是一个诅咒。然后，由于溶液中柔性大分子的IDy，每个给受体对之间的距离不断变化，导致kFRET分布不断变化。这些条件导致难以处理的数学和FRET只能定性研究这些复杂的大分子在溶液中的ILSs和IDy。这是一个真正的科学耻辱，因为许多大分子现在可以通过对其复杂结构的精细控制来制备，但其在溶液中的定量研究目前是FRET无法达到的。

Fluorescence resonance energy transfer (FRET) is certainly the best known fluorescence technique to probe internal length scales (ILSs) and internal dynamics (IDy) of macromolecules in solution. These parameters matter as they explain what a macromolecule can do. Since macromolecules have numerous industrial applications, their study is of great importance, not only from a basic science perspective, but also to the Canadian economy. The principle of FRET is to act as a spectroscopic ruler. The FRET rate constant (kFRET) depends on the distance between an energy donor and energy acceptor dye. This relationship is of considerable value to measure the size of rigid macromolecules by measuring kFRET between two dyes attached at specific positions of a macromolecule. Unfortunately, it becomes a curse for flexible macromolecules having numerous labeling sites separated by multiple distances. Then the distance between every donor-acceptor pair is constantly changing due to the IDy of the flexible macromolecule in solution resulting in a constantly varying kFRET distribution. These conditions lead to intractable mathematics and FRET only enables qualitative studies of the ILSs and IDy of these complex macromolecules in solution. This is a true scientific shame since numerous macromolecules can now be prepared with exquisite control over their complex architecture, but whose quantitative study in solution is presently out of reach by FRET. Pyrene excimer fluorescence (PEF) is another photophysical phenomenon whereby an excimer is generated through the encounter between an excited and a ground state pyrene dye. Contrary to FRET, PEF occurs on contact which means that the rate constant of PEF (kPEF) does not depend on the distance between two dyes, but rather is directly related to the IDy of the macromolecule and the local concentration [Py]loc of pyrene labels covalently attached to the macromolecule. These features simplify dramatically the mathematics involved in the determination of kPEF and this laboratory has recently demonstrated that kPEF can be determined even for pyrene-labeled macromolecules having the most complex macromolecular architecture. In effect, kPEF yields [Py]loc which depends on the macromolecular size, and thus the ILSs of a macromolecule. Thus PEF yields the same information as FRET, but through much simpler mathematics! This proposal will expand on these recent discoveries by applying PEF to study the ILSs and IDy of polypeptides, polymeric bottle brushes, dendrimers, and polysaccharides in solution. These macromolecules are of great current scientific interest. These research topics offer excellent training opportunities to students interested in gaining expertise in Macromolecular Science.