Probing Macromolecules at the Molecular Level by Pyrene Excimer Fluorescence

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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.