Nanoscale dynamics of individual proteins and peptides
- upper: Model of the HTH motif with fluorescent dyes attached (left: Alexa 488, right: Alexa 647)
- lower: Model of the beta-sheet forming WW domain protein
Single molecule fluorescence spectroscopy (SMS) has emerged as a powerful tool in the study of protein folding and protein/peptide conformational dynamics. The advantage of SMS is its ability to watch the folding of individual molecules, which provides key new insights into the heterogeneity of this process. In contrast to ensemble measurements, SMS promises direct access to information on individual folding pathways, as well as to internal conformation dynamics between conformers in the unfolded and folded state. The major tool used in these studies is Förster resonance energy transfer (FRET), which allows for measurement of distance and distance changes between two specific sites in a protein with sub-nanometer accuracy. A powerful alternative is photo-induced electron transfer (PET), which uses the electron-transfer-induced quenching experienced by some fluorescent dyes when coming in contact with the amino acid tryptophan.
However, a severe limitation of current SMS techniques is the short time period (milliseconds) during which a signal from one and the same molecule can be measured, restricting the length of single-molecule fluorescence trajectories that can be recorded. This limitation is due to the diffusion of the molecule out of the detection volume, and due to photobleaching of the used fluorescent marker. We overcome this limitation by employing two new concepts in combination: (i) The recently developed reducing and oxidizing system (ROXS) for dramatically improving photophysical stability, and (ii) the encapsulation of molecules into nanocontainers. This allows for the observation of long fluorescence trajectories of one and the same molecule, which is crucially important for detecting rare events and obtaining sufficiently large data statistics for individual molecules.
The technique will be applied in the study of conformational dynamics of different proteins including 1.) the ultra-fast folding helix-turn-helix motif of the engrailed homeodomain protein (HTH) and 2.) the beta-sheet motif (WW domain) of the Formin-Binding protein as model systems for building blocks of protein tertiary structure.
Intensity based FRET analysis
FLCS based analysis of interconversion rates:
To extract the interconverison rate between two conforma-tions of the molecule we describe it as a system that can switch between a high-FRET (A) and a low-FRET state (B). The donor may also temporally occupy a dark state (e.g. triplet state). This determines a four state system as shown in the figure on the left. We assume that kST and kTS in the high- and low-FRET conformation are the same and obtain the rate equations of the system as:
With the eigenvalues λ1 = 0, λ2 = kAB + kBA, λ3 = kST + kTS, and λ4 = λ2 + λ3 we expect to observe three exponents in the fast part of the correlation function. The second of these exponent corresponds to the sum of the rate-constants of interest. To obtain the individual rates one has to analyze also the amplitudes of the components. One finds:
