Fluorescence-based methods to study rapid dynamics and conformational flexibility in peptides

Intramolecular collision of polypeptides is the primary step in protein folding, the
dynamics of which is of importance for understanding this fascinating topic. In this thesis
the rapid dynamics and flexibility of several sets of peptides were experimentally
investigated with a fluorescence-based method, where the long-lived, hydrophilic
fluorophore, 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), was employed, which can be
selectively and efficiently quenched by tryptophan (Trp) through contact. An asparagine
derivative, Fmoc-DBO, was synthesized and applied to standard solid-phase peptide
synthesis to obtain DBO/Trp-labeled peptides. The end-to-end collision rates can then be
directly related to the intramolecular quenching of DBO by Trp. [Hudgins, R. R.; Huang,
F.; Gramlich, G.; Nau, W. M. J. Am. Chem. Soc. 2002, 124, 556-564 (Appendix I); Nau,
W. M.; Huang, F.; Wang, X.; Bakirci, H.; Gramlich, G.; Marquez, C. Chimia 2003, 57,
161-167 (Appendix III); Marquez, C.; Huang, F.; Nau, W. M. IEEE Trans. Nanobiosci.
2004, 3, 39-45 (Appendix V)]
This method has been further improved by establishing a dual quencher system, i.e.,
tyrosine (Tyr) was employed as an additional quencher, which can react with DBO upon
contact but with a lower efficiency than Trp. The combination of two probe/quencher
pairs with different quenching efficiency as well as the theoretical results for
intermolecular diffusion allows the extrapolation of the microscopic rate constants for
formation and dissociation of the end-to-end encounter complex even in the absence of
diffusion-controlled quenching. [Nau, W. M.; Huang, F.; Wang, X.; Bakirci, H.;
Gramlich, G.; Marquez, C. Chimia 2003, 57, 161-167 (Appendix III); Huang, F.; Hudgins,
R. R.; Nau, W. M. 2004, Submitted for publication (Appendix VI)]
We first applied this fluorescence-based method to measure the end-to-end collision
rate constants in flexible Gly-Ser peptides with varying length. The results suggest that the
behavior of real peptides deviates significantly from that of the ideal chain model and the
speed limit for protein folding should be faster than that reported previously. [Hudgins, R.
R.; Huang, F.; Gramlich, G.; Nau, W. M. J. Am. Chem. Soc. 2002, 124, 556-564
(Appendix I)]
We also investigated the end-to-end collision rates of another series of peptides
composed of different types of amino acids in the backbone but with identical length. The
experimental results have led to a conformational flexibility scale for amino acids in peptides and suggested that the flexibility of peptides is mainly determined by the atoms
and groups in close proximity to the backbone, while the more remote atoms and groups
have a smaller effect on the peptide dynamics due to their larger conformational space.
[Huang, F.; Nau, W. M. Angew. Chem. Int. Ed. 2003, 42, 2269-2272 (Appendix II);
Huang, F.; Nau, W. M. Res. Chem. Intermed. 2004, submitted for publication (Appendix
VII)]
Further investigations on peptides derived from the N-terminal b-hairpin of
ubiquitin were also carried out. The end-to-end collision rates in these peptides showed
significant dependence on the secondary structure, i.e., the turn segment is much more
flexible than the strand segments, which supports a previous proposal that the b-turn is the
initiator for the formation of the whole b-hairpin. Activation energies for end-to-end
collision of these peptides showed a good agreement with the collision rate constants,
which indicates that the activation energy may also be a measure of the flexibility of
peptides although it is not as sensitive as the collision rate. [Huang, F.; Hudgins, R. R.;
Nau, W. M. 2004, Submitted for publication (Appendix VI)]
Additionally, to get more detailed structural information of our peptides and to
reveal the underlying reasons for the deviation of the experimental length dependence of
end-to-end collision rates from the theoretical prediction, intramolecular fluorescence
resonance energy transfer (FRET) was applied as an independent approach to investigate
the dynamics in peptide chains. Two energy donor/acceptor pairs with small Förster
critical radius, where either naphthalene or Trp serves as energy donor and DBO as energy
acceptor, were employed. Energy transfer between naphthalene and DBO was first
investigated at a very short distance, where DBO and naphthalene were separated by
dimethylsiloxy. It was found that the Dexter mechanism might dominate in this system
due to the close proximity of donor and acceptor, the high flexibility of the tether, and the
nonviscous solvent employed. [Pischel, U.; Huang, F.; Nau, W. M. Photochem. Photobiol.
Sci. 2004, 3, 305-310 (Appendix IV)] However, when naphthalene and DBO were
covalently attached to the opposite ends of peptides and studied in water, control
experiments in the presence of cucurbit[7]uril as an encapsulating host suggested that
FRET was the dominant mechanism, which allowed us to apply the FRET technique to
recover the intramolecular end-to-end distance distribution and diffusion coefficient by
means of global analysis. In the investigation with naphthalene/DBO energy donor/acceptor pair, slower diffusion coefficients in shorter chains were found for the
series of flexible Gly-Ser peptides, suggesting that shorter chains may exhibit a larger
internal friction limiting the conformational change. Additionally, the intramolecular
energy transfer efficiency have been measured with the Trp/DBO pair and the effective
average end-to-end distances were calculated, which provided a lower limit for the mean
end-to-end distance of peptides for the global data analysis and offered a complementary
approach to interpret the end-to-end collision rates determined with the same pair but
based on a collision-induced quenching mechanism. [Huang, F.; Wang, X.; Haas, E.; Nau,
W. M. 2004, In preparation (Appendix VIII)]
The fluorescence-based method based on contact quenching mechanism has some
other potential applications. It has potential to be applied for high-throughput screening of
protease activity and to investigate the helix-coil transition in peptides.