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| Thomas Kiefhaber, University of Basel |
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| Thomas Kiefhaber is Professor of Biophysical Chemistry at the Biozentrum of the University of Basel (Switzerland). He studied Biology at the University of Regensburg (Germany) and, after completing his PhD in Biophysical Chemistry, spent two years as a post-doctoral fellow with R. L. Baldwin at Stanford University. Since 1993 he is a member of the faculty of Basel University and was named professor for Biophysical Chemistry in 1999. Thomas Kiefhaber's research is centered on the investigation of kinetics and mechanism of protein folding with biophysical methods.
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Protein Dynamics Measured by Triplet-Triplet Energy Transfer
Thomas Kiefhaber, Biozentrum der Universität Basel, Division of Biophysical Chemistry, Klingelbergstr. 70, CH-4056 Basel, Switzerland
The rate at which a protein can explore conformational space during folding is limited by intrachain diffusion processes. The maximum rate for protein folding can not exceed the rates of intramolecular contact formation [1]. To understand the dynamics of unfolded polypeptide chains we studied contact formation between individual amino acid residues using diffusion controlled triplet-triplet energy transfer (TTET) [2]. We attached xanthone as triplet donor and naphtylalanine as triplet acceptor at the ends of polypeptide chains with different sequence and varied the distance between donor and acceptor from 3 to 60 amino acids. We observed single exponential kinetics for end-to-end diffusion in all peptides. In flexiblepoly(Gly-Ser) chains the time constant for contact formation reaches an upper limit of about 5 ns, which sets the speed limit for protein folding. This value is nearly independent of chain length for short donor-acceptor distances (N<8; N=number of peptide bonds between donor and acceptor). In the limit of long chains (N>20) the rate of end-to-end diffusion scales with N^(-1.7). We found only little effect of the amino acid sequence on local chain dynamics. In host-guest peptides the stiffest side chain (Pro) showed only about 8-fold slower chain dynamics compared to the most flexible amino acid (Gly) [3].
During protein folding most interactions are formed between amino acids in the interior of the polypeptide chain. To test for differences in the dynamics of intrachain diffusion compared to end-to-end diffusion we studied contact formation in a series of peptides that had either one end or both ends extended beyond the position of the triplet donor/acceptor. We observed significantly decreased contact rates with increasing mass/surface of the additional tails until a limiting value is reached.
To compare the results obtained from homo-polypetides with natural protein sequences we measured dynamics in various loops from carp-parvalbumin and in the GB1 hairpin [4]. The results are in agreement with the dynamics expected from the host-guest peptides. Interestingly, intrachain diffusion in the natural sequences showed significant activation energies, which were similar to activation energies found for protein folding reactions (15-20 kJ/mol). Studies on helical peptides further revealed that TTET can be used to measure unfolding dynamics of protein secondary structures.
References:
1. Bieri, O., Kiefhaber, T., Biol. Chem. 380 (1999) 923.
2. Bieri, O., Wirz, J., Hellrung, B., Schutkowski, M., Drewello, M., Kiefhaber, T., Proc.
Natl. Acad. Sci. USA 96 (1999) 9597.
3. Krieger, F., Fierz, B., Bieri, O., Drewello, M., Kiefhaber, T., J. Mol. Biol. 332 (2003)
265.
4. Krieger, F., Fierz, B., Axthelm, F., Joder, K., Meyer, D., Kiefhaber, T., Chemical
Physics (2004) in press.
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