TY - JOUR
T1 - Resolution of two sub-populations of conformers and their individual dynamics by time resolved ensemble level FRET measurements
AU - Rahamim, Gil
AU - Chemerovski-Glikman, Marina
AU - Rahimipour, Shai
AU - Amir, Dan
AU - Haas, Elisha
N1 - Publisher Copyright:
© 2015 Rahamim et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - 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 suchmeasurements 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 halfmaximum (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 flexiblemolecule 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 ofmixtures of polypeptides recovered two endto-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 flexiblemacromolecules.
AB - 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 suchmeasurements 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 halfmaximum (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 flexiblemolecule 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 ofmixtures of polypeptides recovered two endto-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 flexiblemacromolecules.
UR - http://www.scopus.com/inward/record.url?scp=84956900874&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0143732
DO - 10.1371/journal.pone.0143732
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C2 - 26699718
AN - SCOPUS:84956900874
SN - 1932-6203
VL - 10
JO - PLoS ONE
JF - PLoS ONE
IS - 12
M1 - e0143732
ER -