Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jul 31;390(5):1074-85.
doi: 10.1016/j.jmb.2009.05.010. Epub 2009 May 13.

Different members of a simple three-helix bundle protein family have very different folding rate constants and fold by different mechanisms

Beth G Wensley  1 , Martina Gärtner, Wan Xian Choo, Sarah Batey, Jane Clarke
Affiliations

Different members of a simple three-helix bundle protein family have very different folding rate constants and fold by different mechanisms

Beth G Wensley et al. J Mol Biol. .

Abstract

The 15th, 16th, and 17th repeats of chicken brain alpha-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected. The rate-limiting transition state for R15 folding is investigated using protein engineering methods (Phi-value analysis) and compared with previously completed analyses of R16 and R17. Characterisation of many mutants suggests that all three proteins have similar complexity in the folding landscape. The early rate-limiting transition states of the three domains are similar in terms of overall structure, but there are significant differences in the patterns of Phi-values. R15 apparently folds via a nucleation-condensation mechanism, which involves concomitant folding and packing of the A- and C-helices, establishing the correct topology. R16 and R17 fold via a more framework-like mechanism, which may impede the search to find the correct packing of the helices, providing a possible explanation for the fast folding of R15.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Kinetics of wild-type spectrin domains. (a) The three wild-type spectrin domains at 25 °C. R15 (black) (un)folds approximately 3 orders of magnitude faster than R16 (red) and R17 (blue). No data were collected when the rate constant exceeded 660 s− 1, as this is the limit of accuracy of our stopped-flow instruments. The continuous black line represents the fit of the R15 data to a standard two-state folding model, aiding the comparison of R15 with R16 and R17. (b) Wild-type R15 at 10 °C. No data are included where the rate constants are < 660 s− 1, and no data are collected at urea concentrations over 7 M due to potential mixing artifacts caused by viscous solutions at low temperatures. The continuous line again represents the fit of the data to a standard two-state folding model. The folding and unfolding arms of R15 that are accessible at 10 °C are longer than those at 25 °C, but are still relatively short compared with those of R16 and R17. Data for (a) were taken from Scott et al.
Fig. 2
Fig. 2
Chevron plots and fits for core mutants. (a) Core mutants in helix A; (b) core mutants in helix B; (c) core mutants in helix C. Continuous lines represent the fit for each mutant to a globally fitted two-state fit with a shared mkf.
Fig. 3
Fig. 3
Altering the helical propensity of R16. Helical propensities calculated at 50 mM ionic strength and 25 °C using AGADIR. (a) Helical propensity of R15 (black), R16 (red), and R17 (blue). The mean values are 2.1% for R15, 6.3% for R16, and 3.9% for R17. (b) Helical propensities of R16rh (purple) and R16ih (green). The mean values are 1.4% for R16rh and 6.9% for R16ih. (c) Chevron plots at 25 °C for wild-type R16 (black), R16rh (purple), and R16ih (green). Alterations in helical propensity do not speed the folding of R16.
Fig. 4
Fig. 4
Comparison of the Φ-values of R15 and R16. (a) Histograms of Φ-values of the rate-limiting transition state at low denaturant concentrations for R15 (Φf; top) and R16 (Φearly; bottom15). Core mutants are shown in dark blue, and exposed Ala-Gly mutations are shown in pale blue. (b–d) Ribbon diagrams showing the R15 Φf-values mapped onto the structure of R15 (from PDB file 1u4q18). (b) The A-helix–C-helix interface and (c) the B-helix. (d) The same structure as in (b) showing core mutations as space-filling models. (e) Ribbon diagram showing the R16 Φearly-values mapped onto the R16 structure (from PDB file 1u4q), showing the A-helix–C-helix interface and core mutations presented as space-filling models. Low Φ-values (0.0–0.3) are shown in red, medium Φ-values (0.3–0.6) are shown in purple, and high Φ-values (0.6–1.0) are shown in blue.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gunasekaran K., Eyles S.J., Hagler A.T., Gierasch L.M. Keeping it in the family: folding studies of related proteins. Curr. Opin. Struct. Biol. 2001;11:83–93. - PubMed
    1. Zarrine-Afsar A., Larson S.M., Davidson A.R. The family feud: do proteins with similar structures fold via the same pathway? J. Mol. Biol. 2005;15:42–49. - PubMed
    1. Gianni S., Guydosh N.R., Khan F., Caldas T.D., Mayor U., White G.W. Unifying features in protein-folding mechanisms. Proc. Natl Acad. Sci. USA. 2003;100:13286–13291. - PMC - PubMed
    1. Religa T.L., Johnson C.M., Vu D.M., Brewer S.H., Dyer R.B., Fersht A.R. The helix–turn–helix motif as an ultrafast independently folding domain: the pathway of folding of engrailed homeodomain. Proc. Natl Acad. Sci. USA. 2007;104:9272–9277. - PMC - PubMed
    1. Religa T.L., Markson J.S., Mayor U., Freund S.M., Fersht A.R. Solution structure of a protein denatured state and folding intermediate. Nature. 2005;437:1053–1056. - PubMed

Publication types

LinkOut - more resources