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