Arborescent Poly(l‑glutamic acid)s as Standards To Study the Dense Interior of Polypeptide Mesoglobules by Pyrene Excimer Fluorescence

A series of arborescent poly­(l-glutamic acid)­s of generations 0 to 3 (PGA­(GY) with Y = 0–3) were randomly labeled with 1-pyrenemethylamine to yield several Py-PGA­(GY) constructs with pyrene contents ranging between 2.5 and 22 mol %. The density (ρ) of the interior of the PGA­(GY) samples was estimated in N,N-dimethyl­formamide (DMF) and dimethyl sulfoxide (DMSO) by conducting gel permeation chromatography and dynamic light scattering experiments to determine their molar mass and hydrodynamic diameter, respectively. It was determined that ρ increased with the generation number from PGA­(G1) to PGA­(G2), which promoted more contacts between the pyrene labels. The increase in the number of pyrene–pyrene contacts was quantified with the parameter N blob obtained by analysis of the fluorescence decays for the Py-PGA­(GY) samples in DMF and DMSO. In the analysis, N blob represented the number of structural units, i.e., glutamic acid residues, comprised inside the volume probed by an exited pyrene, termed a blob. Inside a blob, pyrene excimer formation (PEF) could occur upon diffusive encounters between an excited- and a ground-state pyrene label. It was found that N blob increased with the generation number, and larger N blob values were retrieved in DMSO as compared to DMF because the oligo­(l-glutamic acid) (OGA) side chains in the PGA­(GY) samples underwent partial loss of helicity in DMSO, which increased their hydrodynamic volume and forced the side chains closer to each other, thus resulting in more pyrene–pyrene contacts and larger N blob values. The trends observed for N blob in DMF as a function of the generation number could be correlated theoretically with the degree of polymerization of the OGA side chains used to prepare the PGA­(GY) samples and their internal density ρ. The good agreement found between the theoretical and experimental N blob values confirms that pyrene is an excellent probe to study the complex interior of partially structured polypeptides.