Takuhiro Otosu

Microsecond protein dynamics observed at the single-molecule level

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

Multiple conformational state of human serum albumin around single tryptophan residue at various pH revealed by time-resolved fluorescence spectroscopy

Human serum albumin (HSA) plays important roles in transport of fatty acids and binding a variety of drugs and organic compounds in the circulatory system. This protein experiences several conformational transitions by the change of pH, and the resulting conformations were essential for completing the physiological roles in vivo. Steady-state and time-resolved fluorescence spectroscopy was applied to single tryptophan residue solely arranged in HSA to study subtle conformational change around single tryptophan residue in HSA at various pH. The results showed the characteristic feature of local conformation around tryptophan residue in domain II responding to the change in entire structure. The study of time-resolved area-normalized fluorescence emission spectra (TRANES) also showed the peculiar dielectric property of water molecule trapped nearby tryptophan residue depending on pH. These results suggested that microenvironment around tryptophan residue was tightly packed at acidic and basic pH although entire conformation was loosened.

Fluorescence decay characteristics of indole compounds revealed by time-resolved area-normalized emission spectroscopy.

Time-resolved fluorescence spectroscopy of tryptophan residue has been extensively applied to the studies on structure-function relationships of protein. Regardless of this, the fluorescence decay mechanism and kinetics of tryptophan residue in many proteins still remains unclear. Previous studies have demonstrated that conformational heterogeneity and relaxation dynamics are both involved in the peculiar multiexponential decay kinetics in subnanosecond resolution. In the present study, we characterized the fluorescence decay property of six indole compounds in glycerol by resolving the contribution of conformational heterogeneity and relaxation dynamics. We applied the time-resolved area-normalized fluorescence emission spectrum (TRANES) method for the fluorescence decay analysis. The results of TRANES, time-dependent shift of fluorescence spectral center of gravity, and fluorescence decay simulation demonstrated that the dielectric relaxation process independent of intrinsic rotamer/conformer and the individual fluorescence lifetime gives the peculiarity to the fluorescence decay of indole compounds. These results confirmed that TRANES and time-dependent spectral shift analysis are potent methods to resolve the origin of multiexponential decay kinetics of tryptophyl fluorescence in protein.

The Unfolding of α-Momorcharin Proceeds Through the Compact Folded Intermediate

The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2-3 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state.

Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis

The counting-rate dependence of the temporal response of single photon avalanche diodes (SPADs) is a critical issue for the accurate determination of the fluorescence lifetime. In this study, the response of SPADs was examined with analyzing the time interval of the detected photons. The results clearly show that the shift of the detection timing causes the counting-rate dependence of the temporal response, and this timing shift is solely determined by the time interval from the preceding photon. We demonstrate that this timing instability is readily calibrated by utilizing the macrotime data taken with the time-tag mode that is implemented in the time-correlated single photon counting modules.

Lifetime-Weighted FCS and 2D FLCS: Advanced Application of Time-Tagged TCSPC

Time-tagged TCSPC (time-correlated single photon counting) is a special acquisition mode of TCSPC with which one determines not only the excitation-emission delay time of detected photons but also their arrival times measured from the start of the experiment. Time-tagged TCSPC enables us to examine slow fluctuation of fluorescence lifetimes, which is particularly important in the study of heterogeneous or fluctuating systems at the single-molecule level. In this chapter, we describe recent development of new methods using time-tagged TCSPC, aiming at showing their high potential in studying dynamics of complex systems. We depict two closely related methods based on fluorescence correlation spectroscopy (FCS), i.e., lifetime-weighted FCS and two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). These methods enable us to quantify fluorescence lifetime fluctuations on the microsecond timescale. Showing examples including the study of a biological macromolecule, we demonstrate the usefulness of these two methods in real applications. In addition, we present another application of time-tagged TCSPC, which analyzes photon interval time for characterizing timing instability of photon detectors.

Heterogeneous Packing in the Folding/Unfolding Intermediate State of Bitter Gourd Trypsin Inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.