Interrupted Hydrogen/deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of Alpha-beta Parallel Protein Flavodoxin

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2009 Dec 5

PMID 19959481

Citations 7

Authors

Sanne M Nabuurs

Carlo P M van Mierlo

Affiliations

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Abstract

Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of alpha-beta parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no beta-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe(44) for Tyr(44). To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu(110)-Val(125) is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu(110)-Val(125) thus form the stable core of the helical molten globule of alpha-beta parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel beta-sheet of native flavodoxin. Hence, to produce native alpha-beta parallel protein molecules, the off-pathway species needs to unfold.

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References

Nabuurs S, Westphal A, Aan den Toorn M, Lindhoud S, van Mierlo C . Topological switching between an alpha-beta parallel protein and a remarkably helical molten globule. J Am Chem Soc. 2009; 131(23):8290-5. DOI: 10.1021/ja9014309. View

Dill K, Chan H . From Levinthal to pathways to funnels. Nat Struct Biol. 1997; 4(1):10-9. DOI: 10.1038/nsb0197-10. View

Nabuurs S, Westphal A, van Mierlo C . Noncooperative Formation of the off-pathway molten globule during folding of the alpha-beta parallel protein apoflavodoxin. J Am Chem Soc. 2009; 131(7):2739-46. DOI: 10.1021/ja8089476. View

Redfield C . Using nuclear magnetic resonance spectroscopy to study molten globule states of proteins. Methods. 2004; 34(1):121-32. DOI: 10.1016/j.ymeth.2004.03.009. View

Engel M, Visser A, van Mierlo C . Conformation and orientation of a protein folding intermediate trapped by adsorption. Proc Natl Acad Sci U S A. 2004; 101(31):11316-21. PMC: 509200. DOI: 10.1073/pnas.0401603101. View

Nishimura C, Dyson H, Wright P . Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments. Proc Natl Acad Sci U S A. 2005; 102(13):4765-70. PMC: 555694. DOI: 10.1073/pnas.0409538102. View

Hughson F, Wright P, Baldwin R . Structural characterization of a partly folded apomyoglobin intermediate. Science. 1990; 249(4976):1544-8. DOI: 10.1126/science.2218495. View

Bryngelson J, Onuchic J, Socci N, Wolynes P . Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins. 1995; 21(3):167-95. DOI: 10.1002/prot.340210302. View

Kato H, Feng H, Bai Y . The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate. J Mol Biol. 2006; 365(3):870-80. PMC: 2494534. DOI: 10.1016/j.jmb.2006.10.047. View

10.

Jennings P, Wright P . Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science. 1993; 262(5135):892-6. DOI: 10.1126/science.8235610. View

11.

van Mierlo C, van Dongen W, Vergeldt F, van Berkel W, Steensma E . The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate. Protein Sci. 1998; 7(11):2331-44. PMC: 2143863. DOI: 10.1002/pro.5560071110. View

12.

Jahn T, Radford S . Folding versus aggregation: polypeptide conformations on competing pathways. Arch Biochem Biophys. 2007; 469(1):100-17. PMC: 2706318. DOI: 10.1016/j.abb.2007.05.015. View

13.

Steensma E, van Mierlo C . Structural characterisation of apoflavodoxin shows that the location of the stable nucleus differs among proteins with a flavodoxin-like topology. J Mol Biol. 1998; 282(3):653-66. DOI: 10.1006/jmbi.1998.2045. View

14.

Fernandez-Recio J, Genzor C, Sancho J . Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate. Biochemistry. 2001; 40(50):15234-45. DOI: 10.1021/bi010216t. View

15.

Nabuurs S, Westphal A, van Mierlo C . Extensive formation of off-pathway species during folding of an alpha-beta parallel protein is due to docking of (non)native structure elements in unfolded molecules. J Am Chem Soc. 2008; 130(50):16914-20. DOI: 10.1021/ja803841n. View

16.

Forge V, Wijesinha R, Balbach J, Brew K, Robinson C, Redfield C . Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin. J Mol Biol. 1999; 288(4):673-88. DOI: 10.1006/jmbi.1999.2687. View

17.

Raschke T, Marqusee S . The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions. Nat Struct Biol. 1997; 4(4):298-304. DOI: 10.1038/nsb0497-298. View

18.

Bollen Y, Kamphuis M, van Mierlo C . The folding energy landscape of apoflavodoxin is rugged: hydrogen exchange reveals nonproductive misfolded intermediates. Proc Natl Acad Sci U S A. 2006; 103(11):4095-100. PMC: 1449652. DOI: 10.1073/pnas.0509133103. View

19.

Arai M, Kuwajima K . Rapid formation of a molten globule intermediate in refolding of alpha-lactalbumin. Fold Des. 1996; 1(4):275-87. DOI: 10.1016/s1359-0278(96)00041-7. View

20.

Baldwin R . Protein folding. Matching speed and stability. Nature. 1994; 369(6477):183-4. DOI: 10.1038/369183a0. View