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

Abstract

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 Ϸ100 s and distances greater than Ϸ2 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-s 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 temperaturejump (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 phys''). D phys 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 phys 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.fluorescence correlation spectroscopy ͉ intrachain diffusion ͉ photoinduced electron transfer ͉ protein folding ͉ speed limit S ingle-molecule spectroscopy detects molecular heterogeneities that are hidden in the ensemble average of conventional spectroscopy and provides complementary insights into the conformationally heterogeneous process of protein folding (1, 2). Commonly applied single-molecule fluorescence resonance energy transfer (smFRET) methods have limitations in revealing conformational changes of less than Ϸ2 nm or measuring very fast processes occurring in under Ϸ1 ms because of inherently low fluorescence photon statistics and interference from photophysical effects. The investigation of small and ultrafast folding protein domains, which often represent the building blocks of larger assemblies, is of particular importance to our fundamental understanding of folding. Importantly, their time scales of conformational motions can overlap with those of atomistic computer simulations of protein folding pathways (3).Current limitations of smFRET methods can be overcome using new developments in fluorescence correlation spectroscopy (FCS). FCS measures the kinetics of stochastic fluorescence fluctuations arising from individual fluorescent molecul...

Kinetics of chain motions within a protein-folding intermediate

“…Such a kinetic phase was observed previously in infrared (IR) T-jump experiments but remained unassigned (9). The observed ns phase in PET-FCS is a signature of the unfolded state and reflects the rate constant of intrachain contact formation or loop closure, k U , in a coiled protein chain (16,17). Taking into account differences in flexibility of interior compared with terminal chain segments (18), the observed loop closure kinetics resembled those determined for unfolded model peptides of similar size (19).…”

“…Fluorescence correlation spectroscopy (FCS) analyzes fluorescence fluctuations at equilibrium arising from individual molecules diffusing through a confocal detection volume by Brownian motion (14,15). By combining PET fluorescence quenching with FCS we can detect and analyze kinetics of protein chain motions and fast folding at equilibrium by correlation analysis (12,16).…”

Extremely slow intramolecular diffusion in unfolded protein L

“…At low pH, collapse of the aciddenatured protein after T-jump to 300 K occurs in ∼60 ns (23), but in neutral pH a second relaxation occurs in ∼15 μs (24). These two relaxations are also observed by contact quenching (25).…”

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

“…However it is not yet able to determine dissociation constants for proteins, measure the conformation and stability of proteins, or detect and quantify the interaction of a protein with small drug-like molecules or ligands. Greater sensitivity at the single molecule level has been achieved recently for the measurement of conformational changes by fluorescence correlation spectroscopy, though this again relies upon the labeling of proteins with a strong fluorophore such as Alexa or AttoOxa11 dyes, 24,25 or the use of highly fluorescent proteins such as yellow fluorescent protein (YFP). 26 Here we present a microfluidic measurement technique that enables the thermodynamic stability of reversibly unfolding proteins, and the affinity of a small molecule drug compound for a target protein, to be determined using small samples and with high precision.…”