Structure and dynamics of unfolded polypeptide chains

Möglich, Andreas. Structure and dynamics of unfolded polypeptide chains. 2005, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_7341

Downloads: Statistics Overview


This thesis aimed at improving our knowledge about the dynamic and structural properties of the unfolded states of proteins. Unstructured peptides were used as model systems to probe the structure and the dynamics of such states. Special emphasis was put on the influence of chemical denaturants as they are widely used in protein folding and stability studies.
Triplet-triplet energy transfer (TTET) between xanthonic acid and naphthalene occurs in a diffusion-controlled manner and is thus well suited to measure absolute contact formation rate constants. Using TTET, intrachain loop closure dynamics in unstructured peptides were studied. We found that these dynamics are decelerated by the addition of the denaturants urea and guanidinium chloride (GdmCl). Closer inspection revealed that this behaviour has contributions from two separate effects. First, denaturants increase solvent viscosity which impedes motions of the peptide chain. Secondly, denaturants bind to peptides and thereby further slow chain dynamics. Denaturant binding was found to be rather weak and transient. Urea and GdmCl mainly differ in their binding strength but not in their effect on chain dynamics once bound. This implies similar mechanisms of denaturation for these two compounds.
In order to understand the effect of denaturants on the dynamics of unfolded polypeptide chains in more detail, we studied loop closure dynamics in a series of host-guest peptides. Different 'guest' amino acids were incorporated into common 'host' peptide contexts to assess their influence on peptide dynamics. For most peptides the effect of urea and GdmCl on intrachain contact formation was similar, indicating a common mechanism for denaturant action. These findings imply that chemical denaturants mainly interact with the polypeptide backbone. Lysine and arginine displayed significantly weaker interactions with GdmCl than all other residues, which is most likely due to charge-charge repulsion between the guanidinium cation and the basic amino acid sidechain.
to obtain information abotu the structure of unfolded peptides and thus deepen our understanding of their behaviour, we employed fluorescence resonance energy treansfer (FRET) as a second optical spectroscopic technique. Using FRET, we studied the dynamics and dimensions of unstructured peptides at different GdmCl concentrations. The FRET measurements on these unstructured peptides were complicated by fast conformational rearrangements occuring during the experimental observation time. We developed a strategy to accurately account for these dynamics and to reliably analyze the experimental data. In separate experiments two chromophore pairs were used, which had largely different donor fluorescence lifetimes but were sensitive to the same distance range. Data obtained on both systems were globally evaluated and allowed to assess the influence of fast dynamics on the measured FRET signal. In principle, this strategy could also be applied to the study of other flexible systems. The analysis of the FRET data revealed that the end-to-end distance of unstructured peptides increases with increasing denaturant concentration. These results show that unstructured polypeptides can undergo significant changes in their dimensions upon changes of solvent composition. GdmCl further exerted a profound effect on the internal dynamics of peptide chains. High denaturant concentrations facilitated conformational rearrangements of the peptide chain, which was only partly offset by the concomitant increase of solvent viscosity. Taken together, these data confirmed that concentrated GdmCl solutions are good solvents for polypeptides and water is a bad one. Results from FRET and TTET measurements were found to be in good agreement. The denaturant-induced changes in intrachain contact formation rates, average dimensions and internal dynamics of the peptides were clearly correlated. Comparison of the results obtained with the two spectroscopic techniques further showed that the loop closure dynamics of long peptides can be sufficiently described by a theory for polymer dynamics by Szabo et al.
All-atom simulations of sterically allowed peptide conformations were used to rationalize experimental observations on the behaviour of unstructured peptides. Comparison of simulation results with TTET experiments on proline-containing peptides provided an explanation for the different chain dynamics observed for cis- and trans-proline peptide isomers. Further, we compared the simulations to our FRET results. The average dimensions found for simulated peptide conformations closely coresponded to the dimensions experimentally observed for peptides in concentrated GdmCl solutions. Since in the simulations no intramolecular interactions except for excluded volume effects were considered, this could mean that binding of GdmCl to peptides largely abolishes intramolecular interactions. In the absence of these interactions the polypeptide chain would be more extended and able to udnergo faster conformational rearrangements.
A last part of this thesis dealt with the folding reaction of the trimeric foldon domain from T4 phage fibritin. Global evaluation of refolding experiments conducted at different protein concentrations enabled us to determine the minimal folding mechanism. Our data analysis provided an explanation for the high stability and fast assembly of the foldon trimer.
Advisors:Kiefhaber, Thomas
Committee Members:Seelig, Joachim
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Seelig J)
UniBasel Contributors:Seelig, Joachim
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7341
Thesis status:Complete
Number of Pages:179
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 15:20

Repository Staff Only: item control page