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

The denaturation undergone by alpha-helical poly(L-glutamic acid) (PLGA) in N,N-dimethyl-formamide 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 alpha-helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number () of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. remained constant for randomly coiled Py-PDLGA but decreased from similar to 20 to similar to 10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in reflected the decrease in the local density of PLGA as the alpha-helix unraveled in solution. The changes in with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA alpha-helix in DMF. The relationship between and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.

Automated summary

The denaturation process of alpha-helical poly(L-glutamic acid) (PLGA) in N,N-dimethyl-formamide with addition of guanidine hydrochloride (GdHCl) was characterized using fluorescence comparison. By taking advantage of pyrene excimer formation (PEF), changes in the conformation of alpha-helical Py-PLGA were probed. Analysis using Fluorescence Blob Model (FBM) revealed a decrease in the average number of glutamic acids located inside a blob as GdHCl concentration increased, indicating the unravelling of the alpha-helix in PLGA.