The conformation of a series of pyrene-labeled poly(L-lysine)s (Py-PLLs) in 60:40 and 90:10 (v/v) acetonitrile:-water mixtures was determined by comparing the results obtained from the fluorescence blob model (FBM) analysis of their fluorescence decays with those obtained from molecular mechanics optimizations (MMOs). PLL aggregates formed in both solutions as demonstrated by FRET experiments between naphthalene- and pyrene-labeled PLLs. Addition of an excess of unlabeled PLL allowed the conformational study of isolated Py-PLL embedded in a matrix of unlabeled PLLs. By varying the acetonitrile (ACN) content of the solution from 60 to 90 vol % ACN, Py-PLL was found to undergo a conformational change from a random coil to an alpha-helix. The conformational change induced an increase in the maximum number of lysines (N-blob) separating two pyrene-labeled lysines that could still form an excimer between an excited- and a ground-state pyrene. N-blob obtained from the FBM analysis increased from 15.2 +/- 2.1 to 25.2 +/- 1.2 lysines as PLL changed its conformation from a random coil to an alpha-helix. AFM revealed that the alpha-helical PLLs organized themselves into structured bundles similar to 22 nm in diameter. The FBM analysis of the decays acquired with a solution of aggregated Py-PLLs in a 90:10 ACN:water mixture yielded a larger N-blob value of 36.6 +/- 3.4. The increase in N-blob indicated that the Py-PLL constructs could now interact with one another in the helical bundles. This increase in N-blob was then used in conjunction with MMOs to determine an interhelical spacing of 2.9 +/- 0.1 nm for Py-PLLs in a bundle. This interhelical spacing resulted in a local density of 0.25 +/- 0.01 g.cm(-3) for the bundles of PLL alpha-helices, which was a reasonable density for a protein in solution. This study describes an experimental means to probe the number of amino acids that interact with each other as the conformation of a polypeptide evolves from that of a random coil to that of an alpha-helix to finally that of a bundle of alpha-helices.