Effect of Like Charges on the Conformation and Internal Dynamics of Polypeptides Probed by Pyrene Excimer Fluorescence

Abstract

Three series of pyrene-labeled polypeptides, namely, poly­(l-lysine) (Py-PLL), poly­(l-glutamic acid) (Py-PLGA), and poly­(d,l-glutamic acid) (Py-PDLGA), were studied in dimethyl sulfoxide (DMSO) by monitoring their ability to form an excimer between an excited and a ground-state pyrene. The effect that the charges of protonated Py-PLL (Py-PLL·HCl) and deprotonated Py-PLGA (Py-PLGNa) and Py-PDLGA (Py-PDLGNa) had on their conformation and dynamics was assessed by monitoring their fluorescence. The fluorescence decays were analyzed according to the fluorescence blob model (FBM) to determine N blob, which is the number of structural units in a blob, and k blob, which is the rate constant for diffusive encounters between structural units and their side chains inside a blob. FBM analysis indicated that the blob size for Py-PLGA and Py-PDLGA was unaffected by the presence of anionic charges, yielding N blob values of 10.3 ± 1.7 and 18.2 ± 1.1 glutamic acid units, respectively. These N blob values matched the values found for their uncharged counterparts. Molecular mechanics optimizations (MMOs) were then applied to determine the theoretical N blob th value that could be obtained if Py-PLGA adopted the conformation of a random coil, a polyproline type II helix, a 310-helix, or an α-helix. The agreement found between the N blob value of 17.9 ± 1.1 for protonated PLGA and deprotonated PLGNa and the N blob th of 19 found for 310-helical conformation suggested that this was the conformation adopted by the PLGAs in DMSO. Py-PLL·HCl was studied in a similar manner, yielding an N blob value of 14.3 ± 1.3 lysines, which suggested a coiled conformation according to MMOs. Comparison of k blob between charged and neutral polypeptides demonstrated that the presence of charges slowed the dynamics experienced by the amino acids. Because the polypeptide blobs appeared to have features in terms of their size and dynamics that were similar to those of foldons, this study further supports the notion that blobs and foldons might be identical objects.

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

“…As already discussed in earlier works [ 7 , 8 , 9 , 10 , 11 , 12 ], the FBM analysis of the PEF signal generated by macromolecules randomly labeled with pyrene yields the parameter N blob , which describes the conformation of structured macromolecules in solution. The fact that the macromolecule needs to be randomly labeled does not require specific attachment points, and can be polydisperse represents important advantages to the method.…”

“…< N blob > decreased from 20.2 (±1.8) for Py-P L GA in DMF without GdHCl to an average value of 10.2 (±1.5) for Py-P L GA with 4 and 5 M GdHCl. An < N blob > value of 20.2 (±1.8) has been reported numerous times, when Py-P L GA adopts an α–helical conformation [ 9 , 10 , 12 ], as it is known to do in DMF [ 31 ]. With 4 and 5 M GdHCl, < N blob > for Py-P L GA approached the < N blob > value for randomly coiled Py-P DL GA in DMF, that remained constant and equal to 10.4 (±1.3) at all GdHCl concentrations.…”

“…First, pyrene is hydrophobic and aggregates in water, making the structural study of pyrene-labeled macromolecules in water challenging [ 40 ]. Second, the random labeling of a macromolecule is well suited to characterize its structure in solution as long as the pyrenyl labels are attached at its periphery such as onto the side groups of helices of amylose [ 7 ] or P L GA or poly( L -lysine) [ 8 , 9 , 10 , 11 , 12 ]. In the case of a protein containing several closely packed structural motives, the random introduction of pyrenyl labels onto the motives would interfere with their tight packing, which would affect the structure of the protein.…”

“…A repetition rate of 1 MHz or 500 kHz, time per channel of 1.02 or 2.04 ns/ch, and number of counts at the decay maximum of 40,000 or 20,000 counts were applied to the monomer and excimer fluorescence decays, respectively. These experimental settings were the same as those reported in earlier publications [ 10 , 12 ].…”

“…In turn, this information could be related to the conformation of the macromolecule of interest, a most important research topic in macromolecular science. Based on this insight, a combination of pyrene excimer formation (PEF), FBM, and molecular mechanics optimizations (MMOs) was applied to yield the internal density of arborescent poly( L -glutamic acid)s (P L GAs) [ 6 ], confirm the helical conformation of amylose in dimethylsulfoxide (DMSO) [ 7 ], P L GA in N , N -dimethylformamide (DMF) [ 8 , 9 , 10 ], and poly( L -lysine) in acetonitrile/water mixtures [ 11 ], predict the 3 10 -helical conformation of P L GA in DMSO [ 12 ], introduce the Solution-Cluster model to describe the interior of amylopectin in DMSO [ 13 , 14 ], and provide the first 1:1 direct relationship between the experimental and predicted folding time of proteins [ 15 , 16 ].…”