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

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.

Effects of Glycine on the Local Conformation and Internal Backbone Dynamics of Polypeptides

“…Since proteins are composed of 20 common aa's, the study of the specific contributions that a given aa might have on the IBD and conformational freedom of an unfolded protein would be impossible to discern among the contributions of the 19 other aa's. The PEF studies on pyrene-labeled polypeptides described in this and earlier reports 13,14,[26][27][28]35,36,38 illustrate how this information can now be accessed by preparing copolypeptides to gauge the effect of a given comonomer on the homopolymers. The hope is that the combination of the information retrieved from these experiments will provide a simple broad set of rules leading to a better understanding of how proteins fold in solution.…”

“…As suggested earlier, the similar ⟨ k blob × N blob ⟩ product and the relatively narrow range of N blob values between 10 and 22 aa’s expected for any aa sequence suggest that the IBD of unfolded polypeptides are much simpler to understand compared to the much more complex structures that polypeptides can adopt in solution. Furthermore, the PEF-based methodology developed in this and earlier reports ,,− , allows the experimentalist to assess the effects that a specific aa has on the IBD of a polypeptide. Since the folding of proteins is a consequence of their IBD, the study of the IBD of polypeptides by the combination of PEF and FBM analyses described in this report represents a new avenue of research that is expected to provide insights into the complex problem of protein folding.…”

“…Consequently, each blob contains a specific number N blob of structural units, which depends on the conformational freedom of the polymer. 25,26 Polymers with rigid and inflexible backbones result in small blobs containing chain segments that are relatively elongated over the short length-scale of a blob, 26,27 leading to small N blob values. On the other hand, flexible polymers have more coiled conformations within a blob, leading to larger N blob values.…”

“…Instead, the doubling in ⟨N blob ⟩ and halving of ⟨k blob ⟩ over the ca. 3 nm length-scale expected for a PGlu blob 27 suggested that, unlike other aa's, 26 Gly afforded sufficient conformational freedom to increase the local mobility of the polypeptide backbone, thereby generating larger blobs. Despite the significant changes in blob size, the IBD of the polypeptides were hardly affected by Gly, particularly at low Gly contents, where the increase in ⟨N blob ⟩ was balanced by a concomitant increase in V blob , resulting in PGlyGlu having the same ⟨k blob × N blob ⟩ value of 0.14 ns −1 as PGlu.…”

“…A number of studies have demonstrated that the fluorescence blob model (FBM) is a powerful experimental tool to probe the IBD of macromolecules in general − and polypeptides in particular. , The FBM compartmentalizes a polymer chain that has been randomly labeled with the dye pyrene into a string of blobs of equal volume V blob , where V blob is the subvolume of the polymer coil probed by an excited pyrene covalently attached to the chain. Consequently, each blob contains a specific number N blob of structural units, which depends on the conformational freedom of the polymer. , Polymers with rigid and inflexible backbones result in small blobs containing chain segments that are relatively elongated over the short length-scale of a blob, , leading to small N blob values. On the other hand, flexible polymers have more coiled conformations within a blob, leading to larger N blob values .…”