Advantages of Pyrene Excimer Formation (PEF) over Fluorescence Resonance Energy Transfer (FRET) for Probing the Conformation of Macromolecules in Solution

Pyrene excimer formation (PEF) was applied to study the homopolypeptides poly(d,l-glutamic acid) (PDLGA) and poly(l-glutamic acid) (PLGA), collectively referred to as the PGA samples, in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). The polypeptides were labeled with 1-pyrenebutylamine (PyC4N) or 1-pyreneoctylamine (PyC8N) and were described as PyCX(x)N-PGA where X equals 4 or 8, respectively, and x represents the mole percentage of pyrene-labeled glutamic acid (Glu). The fluorescence results obtained with these polypeptides were compared with the results obtained earlier with the same polypeptides randomly labeled with 1-pyrenemethylamine (PyC1N). Analysis of the fluorescence decays of the PyCX(x)N-PGA samples with the fluorescence blob model (FBM) yielded the average number (⟨Nblobexp⟩) of Glu units per blob, where a blob is the volume probed by an excited pyrenyl label. The ⟨Nblobexp⟩ values obtained for the PyCX(x)N-PGA samples in DMF and DMSO were compared with the NblobMMO values determined via molecular mechanics optimizations (MMO) conducted for PLGA constructs adopting four different conformations and labeled with either PyC1N, PyC4N, or PyC8N. The good agreement obtained between the ⟨Nblobexp⟩ and NblobMMO values for PDLGA in DMF and DMSO and PLGA in DMF, whose conformation was known to be a random coil and an α–helix, respectively, validated the PEF-based methodology. It was then applied to determine the unknown conformation of PLGA in DMSO. The excellent match found between ⟨Nblobexp⟩ obtained for PLGA in DMSO and NblobMMO obtained for a PLGA 310 helix led to its unambiguous assignment as the conformation of PLGA in DMSO. Since PyC8N probed a volume whose 7.7 nm diameter was almost double that of PyC1N, these experiments significantly extended the range of distances over which PEF can be applied to characterize macromolecular conformations, making PEF competitive with the distance range accessible by fluorescence resonance energy transfer (FRET).