Quantitative Single-Molecule Conformational Distributions:  A Case Study with Poly-(l-proline)

Precise measurement of the potential of mean force is necessary for a fundamental understanding of the dynamics and chemical reactivity of a biological macromolecule. The unique advantage provided by the recently developed constant-information approach to analyzing time-dependent single-molecule fluorescence measurements was used with maximum entropy deconvolution to create a procedure for the accurate determination of molecular conformational distributions, and analytical expressions for the errors in these distributions were derived. This new method was applied to a derivatized poly(l-proline) series, P_nCG₃K(biotin) (n = 8, 12, 15, 18, and 24), using a modular, server-based single-molecule spectrometer that is capable of registering photon arrival times with a continuous-wave excitation source. To account for potential influence from the microscopic environment, factors that were calibrated and corrected molecule by molecule include background, cross-talk, and detection efficiency. For each single poly(l-proline) molecule, sharply peaked Förster type resonance energy transfer (FRET) efficiency and distance distributions were recovered, indicating a static end-to-end distance on the time scale of measurement. The experimental distances were compared with models of varying rigidity. The results suggest that the 23 Å persistence length wormlike chain model derived from experiments with high molecular weight poly(l-proline) is applicable to short chains as well.