Abstract
Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET) measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET) measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM), and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser)7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.
Highlights
The native conformations of proteins are stabilized by a complex network comprising a very large number of interactions, which leads to a narrow ensemble of conformations, as opposed to the unfolded state and partially folded state ensembles that include a large number of nonnativePLOS ONE | DOI:10.1371/journal.pone.0143732 December 23, 2015time resolved Förster resonance energy transfer (FRET) (trFRET) Resolution of Conformer Subpopulation and highly flexible conformations [1,2,3]
We simulated and analyzed trFRET data corresponding to each sub-population separately, and recovered the parameters of each one of the synthesized distributions
While the returned values of the first sub-population were with a narrow error range, the error range obtained for the full width at half maximum (FWHM) value of the second sub-population was too large
Summary
TrFRET Resolution of Conformer Subpopulation and highly flexible conformations [1,2,3] Characterizing such ensembles requires statistical representations i.e. distributions of distances, their mean, variance, and rates of fast fluctuations. In a two-state protein folding transition [10, 11], the sub-population of very flexible unfolded molecules folds to a less flexible native state. In this kinetic process, the distance distribution parameters of each sub-populations remain constant, while only the ratio between the two subpopulations is shifted [12]
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