Unfolding of Helical Poly(L-Glutamic Acid) in N,N-Dimethylformamide Probed by Pyrene Excimer Fluorescence (PEF)

The denaturation undergone by α–helical poly(L-glutamic acid) (PLGA) in N,N-dimethylformamide upon addition of guanidine hydrochloride (GdHCl) was characterized by comparing the fluorescence of a series of PLGA constructs randomly labeled with the dye pyrene (Py-PLGA) to that of a series of Py-PDLGA samples prepared from a racemic mixture of D,L-glutamic acid. The process of pyrene excimer formation (PEF) was taken advantage of to probe changes in the conformation of α–helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number (<N(blob)>) of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. <N(blob)> remained constant for randomly coiled Py-PDLGA but decreased from ~20 to ~10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in <N(blob)> reflected the decrease in the local density of PLGA as the α–helix unraveled in solution. The changes in <N(blob)> with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α–helix in DMF. The relationship between <N(blob)> and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.