Direct observation of ultrafast folding and denatured state dynamics in single protein molecules Export

@article{citeulike:5978704, abstract = {10.1073/pnas.0910860106 Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than \^{a}‰ˆ100 \^{I}¼s and distances greater than \^{a}‰ˆ2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-\^{I}¼s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (\^{a}€œD\^{a}€). D had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the \^{a}€œspeed limit\^{a}€ of folding. D exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.}, author = {Neuweiler, Hannes and Johnson, Christopher M. and Fersht, Alan R.}, citeulike-article-id = {5978704}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0910860106}, citeulike-linkout-1 = {http://www.pnas.org/content/0910860106v1/.abstract}, citeulike-linkout-2 = {http://www.pnas.org/content/0910860106v1/.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/19841261}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=19841261}, day = {3}, doi = {10.1073/pnas.0910860106}, journal = {Proceedings of the National Academy of Sciences}, keywords = {dynamics, fcs, protein}, month = {November}, number = {44}, pages = {18569--18574}, posted-at = {2009-10-21 16:19:40}, priority = {2}, title = {Direct observation of ultrafast folding and denatured state dynamics in single protein molecules}, url = {http://dx.doi.org/10.1073/pnas.0910860106}, volume = {106}, year = {2009} }

TY - JOUR ID - citeulike:5978704 L3 - citeulike-article-id:5978704 N2 - 10.1073/pnas.0910860106 Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than ≈100 μs and distances greater than ≈2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-μs decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (“D”). D had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the “speed limit” of folding. D exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding. IS - 44 JF - Proceedings of the National Academy of Sciences EP - 18574 TI - Direct observation of ultrafast folding and denatured state dynamics in single protein molecules VL - 106 SP - 18569 KW - dynamics KW - fcs KW - protein AU - Neuweiler, Hannes AU - Johnson, Christopher AU - Fersht, Alan PY - 2009/11/03/ UR - http://dx.doi.org/10.1073/pnas.0910860106 ER -