Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca2+ and Mg2+, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg2+]. Although binding of CoreTet to DNA becomes markedly weaker at higher [Mg2+], binding of TetCT depends slightly on [Mg2+]. Single-molecule fluorescence measurements revealed that the one-dimensional diffusion of CoreTet along DNA consists of fast and slow search modes, the ratio of which depends strongly on [Mg2+]. In contrast, diffusion of TetCT consisted of only the fast mode. The disordered C-terminal domain can associate with DNA irrespective of [Mg2+], and can maintain an equilibrium balance of the two search modes and the p53 search distance. These results suggest that p53 modulates the quaternary structure of the complex between p53 and DNA under different [Mg2+] and that it maintains the target search along DNA.

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins.

The equilibrium unfolding transition of the B domain of protein A (BdpA) was investigated by using single-molecule fluorescence spectroscopy based on line-confocal detection of fast-flowing samples. The method achieved the time resolution of 120 μs and the observation time of a few milliseconds in the single-molecule time-series measurements of fluorescence resonance energy transfer (FRET). Two samples of BdpA doubly labeled with donor and acceptor fluorophores, the first possessing fluorophores at residues 22 and 55 (sample 1) and the second at residues 5 and 55 (sample 2), were prepared. The equilibrium unfolding transition induced by guanidium chloride (GdmCl) was monitored by bulk measurements and demonstrated that the both samples obey the apparent two-state unfolding. In the absence of GdmCl, the single-molecule FRET measurements for the both samples showed a single peak assignable to the native state (N). The FRET efficiency for N shifts to lower values as the increase of GdmCl concentration, suggesting the swelling of the native state structure. At the higher concentration of GdmCl, the both samples convert to the unfolded state (U). Near the unfolding midpoint for sample 1, the kinetic exchange between N and U causes the averaging of the two states and the higher values of the relative fluctuation. The time series for different molecules in U showed slightly different FRET efficiencies, suggesting the apparent heterogeneity. Thus, the high-speed tracking of fluorescence signals from single molecules revealed a complexity and heterogeneity hidden in the apparent two-state behavior of protein folding.

A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, “flow-and-stop” control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.

•The transition path of small proteins can be explained by the native centric model.•The size of the unfolded proteins in the absence of denaturants is controversial.•The unfolded state of small proteins possesses substates with residual structures.•Even small single domain proteins demonstrate complex folding dynamics.•Large proteins are distinct and show rapid collapse with long-range contacts.Progress in our understanding of the simple folding dynamics of small proteins and the complex dynamics of large proteins is reviewed. Recent characterizations of the folding transition path of small proteins revealed a simple dynamics explainable by the native centric model. In contrast, the accumulated data showed the substates containing residual structures in the unfolded state and partially populated intermediates, causing complexity in the early folding dynamics of small proteins. The size of the unfolded proteins in the absence of denaturants is likely expanded but still controversial. The steady progress in the observation of folding of large proteins has clarified the rapid formation of long-range contacts that seem inconsistent with the native centric model, suggesting that the folding strategy of large proteins is distinct from that of small proteins.

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, Dphys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (Rg) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the Rg value extrapolated from kinetic Rg data to time zero, Rg,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in Rg2 was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated Rg of the minor component of Dphys containing the non-native cis proline isomer (Dphys,cis) to be 25.7 ± 0.6 Å. Moreover, Rg of the major component of Dphys containing the native proline isomer (Dphys,tra) was estimated as 23.9 ± 0.2 Å based on Rg,0. Consequently, both components of the burst-phase intermediate of barnase (Dphys,tra and Dphys,cis) are still largely expanded. It was inferred that Dphys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.

•Folding of βLG involves intermediates with non-native α-helix.•Submillisecond dynamics of βLG folding was observed with CD and SAXS.•Initial collapse occurs irrespective of the contents of non-native α-helices.•Non-native helix around strand A might facilitate long-range contact formation.In the folding of β-lactoglobulin (βLG), a predominantly β-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of βLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200 μs. Time-resolved SAXS showed that the radius of gyration (Rg) of the intermediate at 300 μs was 23.3 ± 0.7 Å, indicating a considerable collapse from the unfolded state having Rg of 35.1 ± 7.1 Å. Further compaction to Rg of 21.2 ± 0.3 Å occurred with a time constant of 28 ± 11 ms. Pair distribution functions showed that the intermediate at 300 μs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar Rg of 23.0 ± 0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of βLG.Download high-res image (69KB)Download full-size image

•How does tumor suppressor p53 regulate binding to target on DNA?•Target binding of p53 on DNA was measured at a single-molecule level.•p53 sliding along the DNA showed many pass events over the target.•TRP was regulated by activating or inactivating mutations.•Target recognition regulation might be a general mechanism in transcription factors.Tumor suppressor p53 binds to the target in a genome and regulates the expression of downstream genes. p53 searches for the target by combining three-dimensional diffusion and one-dimensional sliding along the DNA. To examine the regulation mechanism of the target binding, we constructed the pseudo-wild type (pseudo-WT), activated (S392E), and inactive (R248Q) mutants of p53 and observed their target binding in long DNA using single-molecule fluorescence imaging. The pseudo-WT sliding along the DNA showed many pass events over the target and possessed target recognition probability (TRP) of 7 ± 2%. The TRP increased to 18 ± 2% for the activated mutant but decreased to 0% for the inactive mutant. Furthermore, the fraction of the target binding by the one-dimensional sliding among the total binding events increased from 63 ± 9% for the pseudo-WT to 87 ± 2% for the activated mutant. Control of TRP upon activation, as demonstrated here for p53, might be a general activation mechanism of transcription factors.Download high-res image (89KB)Download full-size image

•Does loss of homeostatic control regulate the functional dynamics of p53?•The sliding dynamics of p53 on DNA was measured at a single-molecule level.•The sliding of p53 on DNA significantly depended on [Mg2 +] or [Ca2 +].•Two sliding modes of p53 with different diffusion coefficients were observed.•The search distance of p53 along DNA is maintained as with [Mg2 +] or [Ca2 +].One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg2 + and Ca2 + after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg2 + or Ca2 +, indicating that the divalent cations accelerate the sliding likely by weakening the DNA–p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg2 + and Ca2 +, which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.Download high-res image (85KB)Download full-size image