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Étude des événements cinétiques initiaux du repliement de l'apomyoglobine.

Abstract : A neo-synthesized polypeptide is able to found its specific pathway upon several accessible ways to its final three-dimensional structure by passing trough partially structured intermediate in a very short time. Collecting data in order to improve our knowledge on this intermediate is crucial, but it's necessary to improve mixing apparatus to be able to record the protein fast events of folding.
The purpose of these thesis was to follow fast protein folding events, in particular apomyoglobin (apoMb), by using ultra fast mixing apparatus. A stopped-flow apparatus with a customized micro-cuve, decreases the mixing time. A bimolecular reaction (Nata & NBS) was used to determine a 400±10 µs dead time, in fluorescence and far UV circular dichroïsm modes. ApoMb is a particularly interesting protein for the study of early protein folding events. The ultra-fast stopped-flow allowed us to follow rapid kinetics (k up to 1500 s-1). The data show that each previously identified steps leading unfolded apoMb to its native conformation, namely the UIa, the IaIb and the IbN reactions, exhibits a two-state highly cooperative behaviour.
We studied the effects of osmolytes on the kinetics and on the equilibrium stability of U, I and N forms of apoMb. Fast kinetic studies in the presence of sucrose, allow to show the behaviour of the UIa reaction. These results show that sucrose destabilizes, the U form and the transition state of the UIa reaction relative to the Ia form. The rate limiting step appears to precede compaction of the peptidic chain. In the same conditions the IbN transition shows transition state characteristics close to those of the Ib state. These results, showing the osmophobic effect on the I intermediate, and also preliminary studies about molecular crowding on cytochrom C, are discussed in these manuscript.
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Contributor : Pascal Martinez <>
Submitted on : Friday, October 12, 2007 - 12:39:40 PM
Last modification on : Friday, November 6, 2020 - 3:45:19 AM
Long-term archiving on: : Sunday, April 11, 2010 - 10:52:59 PM


  • HAL Id : tel-00178851, version 1




Sébastien Weisbuch. Étude des événements cinétiques initiaux du repliement de l'apomyoglobine.. Autre [q-bio.OT]. Université Joseph-Fourier - Grenoble I, 2005. Français. ⟨tel-00178851⟩



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