Rate Limiting Factors For Protein Folding.

Maria Berggård Silow

Research output: ThesisDoctoral Thesis (compilation)


Abstract. This thesis describes factors that are rate limiting for the folding of two small proteins, U1A and CI2 which fold without accumulating intermediates. The [GdnHCl] dependencies of the unfolding- and refolding kinetics of U1A display downward curvatures. However, as the curvatures are precisely matched and no indications of formation of partially structured intermediates are seen, the folding behaviour is still consistent with a two-state model. Instead, the curvatures seem related to Hammond behaviour, i.e. movements of the transition state on the reaction coordinate. This implies that the free energy barrier is broad and rather flat. Consequently the search for productive interactions in the folding protein does not take place at ground state level but at high energy. Analysis of mutants of CI2 shows, that to a larger extent than previously thought, these mutants also display transition state movements. Possibly, the activation barrier for folding is generally broad but with a more or less rugged surface. When the ruggedness involves localised features that are much higher than the surrounding barrier, the protein will appear to have a localised transition state. Mutations that lower these localised features will then set free movements of the transition state. One way to monitor diffusive events in protein folding is to measure the rate of folding as a function of solvent viscosity. Unfortunately the viscogens (osmolytes) added in these studies also tend to stabilise the protein and thus have dual effects on the folding rate. When CI2 is refolded in the presence of several different osmolytes we find no viscosity dependence on the refolding kinetics. However, we find that the osmolytes, in addition to their other effects also induce a collapse of the denatured protein. The collapse increases the reconfiguration time of the protein and thereby retards folding. At [protein]>1mM, U1A aggregates early in kinetic refolding experiments. This leads to a retardation of folding at low [denaturant], which resembles the accumulation of an intermediate. The retardation results from kinetic competition: at high [protein] the fraction of protein that aggregates increases and eventually dominates the refolding amplitude. As the rate of folding after aggregation is slower than direct folding, this results in an apparent decrease of the folding rate. Similar experiments with CI2 show that also this protein undergoes transient aggregation. Furthermore, as both U1A and CI2 fold without accumulating intermediates the protein aggregates likely form directly from the coil.
Original languageEnglish
Awarding Institution
  • Biochemistry and Structural Biology
  • [unknown], [unknown], Supervisor, External person
Award date2000 May 19
ISBN (Print)91-628-4134-3
Publication statusPublished - 2000

Bibliographical note

Defence details

Date: 2000-05-19
Time: 10:15
Place: Sal B Chemical Centre.

External reviewer(s)

Name: Radford, Sheena E.
Title: [unknown]
Affiliation: University of Leeds, UK.


Article: I Silow, M. and Oliveberg, M. (1997). High Energy Channelling in Protein Folding. Biochemistry. June 24;36(25); 7633-7637

Article: II Oliveberg, M., Tan, Y-J., Silow, M. and Fersht, A.R. (1998). The Changing Nature of the Protein Folding Transition State: Implications for the Shape of the Free-energy Profile for Folding. J. Mol. Biol. Apr 10;277(4): 933-943.

Article: III Silow, M. and Oliveberg, M. Osmolyte Induced Coil-Collapse in Protein Folding. Manuscript in preparation.

Article: IV Silow, M. and Oliveberg, M. (1997). Transient Aggregates in Protein Folding are Easily Mistaken for Folding Intermediates. Proc. Natl. Acad. Sci. U.S.A. Jun 10; 94(12): 6084-6086

Article: V Silow, M, Tan, Y-J., Fersht, A.R., and Oliveberg, M. (1999). Formation of Short-Lived Protein Aggregates Directly from the Coil in Two-State Folding. Biochemistry. Oct. 5; 38(40): 13006-13012.

Subject classification (UKÄ)

  • Biological Sciences

Free keywords

  • kinetic competition
  • transient aggregation
  • diffusion control
  • reconfiguration time
  • collapse
  • osmolytes
  • broad barriers
  • Hammond behaviour
  • two-state
  • U1A
  • CI2
  • Proteins
  • enzymology
  • Proteiner
  • enzymologi


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