| 1. |
- Dalby, Paul A, et al.
(författare)
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Folding intermediates of wild-type and mutants of barnase. I. use of @f-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium
- 1998
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 276:3, s. 625-646
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Tidskriftsartikel (refereegranskat)abstract
- It is difficult to determine whether transient folding intermediates have a cooperative (or first-order) folding transition without measuring their rates of formation directly. An intermediate I could be formed by a second-order transition from a denatured state D that is progressively changed into I as conditions are changed. We have not been able to monitor the rate of formation of the folding intermediate of barnase directly, but have analysed its reactivity and the equilibrium constant for its formation over a combination of wide ranges of temperature, concentration of denaturant and structural variation. Phase diagrams have been constructed for wild-type and 16 mutant proteins to map out the nature of the energy landscape of the denatured state. The free energy of unfolding of I, @DGD-I, changes with [urea] according to a highly cooperative transition. Further, mD-I(=@d@DGD-I/@d[urea]) for wild-type and several mutants is relatively insensitive to temperature, as would be expected for an intermediate that is formed cooperatively, rather than one that melts out according to a second-order transition. The @f-values for the formation of I change abruptly through the folding transitions rather than have the smooth changes expected for a second-order transition. There is a subset of mutants for which both mD-I and @f-value analysis indicate that a second intermediate becomes populated close to the melting temperatures of the native proteins. The folding intermediate of barnase is, thus, a relatively discrete and compact entity which is formed cooperatively.
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| 2. |
- Danielsson, Jens, et al.
(författare)
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The Pierced Lasso Topology Leptin has a Bolt on Dynamic Domain Composed by the Disordered Loops I and III
- 2020
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:9, s. 3050-3063
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Tidskriftsartikel (refereegranskat)abstract
- Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.
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| 3. |
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| 4. |
- Johnson, Christopher M, et al.
(författare)
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Thermodynamics of Denaturation of Mutants of Barnase with Disulfide Crosslinks
- 1997
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 268:1, s. 198-208
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Tidskriftsartikel (refereegranskat)abstract
- We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (@DCpd), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of @DCpd is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the @DCpd remains unchanged in a destabilised mutant containing a disulfide. The variation in @DCpd may result from an inherent temperature-dependence of @DCpd, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink.
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| 5. |
- Lindberg, Magnus O, et al.
(författare)
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Folding of circular permutants with decreased contact order: general trend balanced by protein stability
- 2001
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 314:4, s. 891-900
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Tidskriftsartikel (refereegranskat)abstract
- To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small β-α-β protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log kf) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na2SO4 or by comparison of the folding kinetics at the transition mid-point, the dependence between log kf and contact order becomes stronger and follows the general correlation for two-state proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding.
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| 6. |
- 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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| 7. |
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| 8. |
- 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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| 9. |
- Otzen, D E, et al.
(författare)
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Burst-phase expansion of native protein prior to global unfolding in SDS
- 2002
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 315:5, s. 1231-40
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Tidskriftsartikel (refereegranskat)abstract
- Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein engineering the structure of an expanded but native-like intermediate that accumulates transiently in the unfolding reaction of the small protein S6 in the presence of SDS. The results show that the SDS micelles attack the native protein in the dead-time of the denaturation experiment, causing an expansion of the hydrophobic core prior to the major unfolding transition. We distinguish two forms of the unfolding intermediate that are correlated with the micellar structure. With spherical micelles, the expansion is seen mainly as a weakening of the interactions which anchor the two -helices to the core of the S6 structure. With cylindrical micelles, prevalent at higher SDS concentrations, the expansion is more global and produces a species which closely resembles the transition-state structure for unfolding in GdmCl. Despite the highly weakened core, the micelle-associated intermediate displays cooperative unfolding, indicating a significant structural plasticity of the species on the native side of the folding barrier in the presence of SDS.
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