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.