| 1. |
- Oliveberg, Mikael, et al.
(författare)
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The Changing Nature of the Protein Folding Transition State: Implications for the Shape of the Free-energy Profile for Folding
- 1998
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 277:4, s. 933-943
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Tidskriftsartikel (refereegranskat)abstract
- According to landscape theory proteins do not fold by localised pathways, but find their native conformation by a progressive organisation of an ensemble of partly folded structures down a folding funnel. Here, we use kinetics and protein engineering to investigate the shape of the free-energy profile for two-state folding, which is the macroscopic view of the funnel process for small and rapidly folding proteins. Our experiments are based mainly on structural changes of the transition state of chymotrypsin inhibitor 2 (CI2) upon destabilisation with temperature and GdnHCl. The transition state ensemble of CI2 is a localised feature in the free-energy profile that is sharply higher than the other parts of the activation barrier. The relatively fixed position of the CI2 transition state on the reaction coordinate makes it easy to characterise but contributes also to overshadow the rest of the free-energy profile, the shape of which is inaccessible for analysis. Results from mutants of CI2 and comparison with other two-state proteins, however, point at the possibility that the barrier for folding is generally broad and that localised transition states result from minor ripples in the free-energy profile. Accordingly, variabilities in the folding kinetics may not indicate different folding mechanisms, but could be accounted for by various degrees of ruggedness on top of very broad activation barriers for folding. The concept is attractive since it summarises a wide range of folding data which have previously seemed unrelated. It is also supported by theory. Consistent with experiment, broad barriers predict that new transition state ensembles are exposed upon extreme destabilisation or radical mutations.
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| 2. |
- Silow, Maria, et al.
(författare)
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Formation of Short-Lived Protein Aggregates Directly from the Coil in Two-State Folding
- 1999
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 38:40, s. 13006-13012
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Tidskriftsartikel (refereegranskat)abstract
- Recent results on the 102 residue protein U1A show that protein aggregation is not always slow and irreversible but may take place transiently in refolding studies on a millisecond time scale. In this study we observe a similar aggregation behavior with the classical two-state protein CI2. Since both U1A and CI2 appear to fold directly from the coil at low protein concentrations, it is likely that the aggregates also form directly from the coil. This is in contrast to the behavior of larger multistate proteins where aggregation occurs in connection to "sticky" intermediates.
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| 3. |
- Tan, Yee-Joo, et al.
(författare)
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The Rate of Isomerisation of Peptidyl-proline Bonds as a Probe for Interactions in the Physiological Denatured State of Chymotrypsin Inhibitor 2
- 1997
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 269:4, s. 611-622
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Tidskriftsartikel (refereegranskat)abstract
- There are four peptidyl-proline bonds in the 64-residue protein chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which has all trans peptidyl-proline bonds, is much faster than that of the slow-folding species, which have one or more cis peptidyl-proline bonds. In CI2, the slow-folding species can be classified into two groups by their rates of refolding, temperature-dependence, pH-dependence and [GdmCl]-dependence of the rate constants and the effect of peptidyl-prolyl isomerase on the rate constants. The replacement of Pro6 by Ala removes one of the slow refolding phases, suggesting that the cis peptidyl-Pro6 conformation is solely responsible for one of the slow-folding species. Pro6 is located in a region of the protein where non-random interactions have been found in a series of N-terminal fragments of CI2 (residues 1 to 13, 1 to 25, 1 to 28 and 1 to 40). In addition, NMR studies on a mutant fragment, (1-40)T3A, have confirmed that this non-native interaction is associated with the bulky side-chain of Trp5. The atypical rate of cis to trans isomerisation of the peptidyl-Pro bond is indicative of the presence of a similar hydrophobic cluster in the physiological denatured state of intact CI2.
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