Abstract
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 (<Nblob>) of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. <Nblob> 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 <Nblob> reflected the decrease in the local density of PLGA as the α–helix unraveled in solution. The changes in <Nblob> with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α–helix in DMF. The relationship between <Nblob> and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.
Highlights
The Fluorescence Blob Model (FBM) was introduced in 1999 as a mathematical tool to extract quantitative information about the internal dynamics of polymers, that had been randomly labeled with the fluorescent dye pyrene [1]
Since values of ~20 and ~10 are those expected for α–helical and randomly coiled Py-poly(L-glutamic acid) (PLGA), respectively [9,10,12], the vs-[guanidine hydrochloride (GdHCl)] trend obtained for Py-PLGA in Figure 4C was taken as evidence that these
FBM experiments reflected the unravelling of the PLGA α–helix as GdHCl was added to the solution
Summary
The Fluorescence Blob Model (FBM) was introduced in 1999 as a mathematical tool to extract quantitative information about the internal dynamics of polymers, that had been randomly labeled with the fluorescent dye pyrene [1]. As discussed in several reviews [2,3,4,5], the FBM takes advantage of the ability of an excited pyrene to form an excimer upon diffusive encounter with a ground-state pyrene inside the volume probed by the excited pyrene, and referred to as a blob. Random labeling of a polymer with a pyrene derivative ensured that the pyrenyl labels would distribute themselves randomly among the blobs according to a Poisson distribution. Analysis of the fluorescence decays acquired with solutions of the pyrene-labeled polymers yielded the average number () of ground-state pyrenes per blob, which could be related to the number (Nblob ) of structural units encompassed inside a blob. A larger Nblob typically indicated that the excited pyrenyl label could probe a larger volume, reflecting a more flexible polymeric backbone
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