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. 2001 Jun;27(2-3):99–117. doi: 10.1023/A:1013137924581

A Comparison of Experimental and Computational Methods for Mapping the Interactions Present in the Transition State for Folding of FKBP12

ERG Main 1, KATE F Fulton 1, VALERIE Daggett 2, SE Jackson 1
PMCID: PMC3456587  PMID: 23345737

Abstract

The folding pathway of FKBP12, a 107 residue α/β protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s-1 at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the α-helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with φ-value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom α-spectrin. For FKBP12, the central three strands of the β-sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central β-strand,makes approximately 60% of its native interactions. The outer β-strands, and the ends of the central β-strands are formed to a lesser degree. The short α-helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within β-strands 2, 4 and 5 and the C-terminus of the α-helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.

Keywords: FKBP12, immunophilin, protein folding, transition state, two-state folding

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References

  • 1.Sancho J., Meiering E., Fersht A.R. Mapping Transition States of Protein Unfolding by Protein Engineering of Ligand-Binding Sites. J. Mol. Biol. 1991;221:1007–1014. doi: 10.1016/0022-2836(91)80188-z. [DOI] [PubMed] [Google Scholar]
  • 2.Chiti F., Taddei N., van Nuland N.A.J., Magherini F., Stefani M., Ramponi G., Dobson C.M. Structural Characterisation of the Transition State for Folding of Muscle Acylphophatase. J. Mol. Biol. 1998;283:893–903. doi: 10.1006/jmbi.1998.2010. [DOI] [PubMed] [Google Scholar]
  • 3.Chiti F., Taddei N., Webster P., Hamada D., Fiaschi T., Ramponi G., Dobson C.M. Acceleration of the Folding of Acylphosphatase by Stabilisation of Local Secondary Structure. Nat. Struct. Biol. 1999;6:380–387. doi: 10.1038/7616. [DOI] [PubMed] [Google Scholar]
  • 4.Fersht A.R., Matouschek A., Serrano L. The Folding of an Enzyme. 1. Theory of Protein Engineering Analysis of Stability and Pathway of Protein Folding. J. Mol. Biol. 1992;224:771–782. doi: 10.1016/0022-2836(92)90561-w. [DOI] [PubMed] [Google Scholar]
  • 5.Daggett V., Li A.J., Itzhaki L.S., Otzen D.E., Fersht A.R. Structure of the Transition-State for Folding of a Protein-Derived from Experiment and Simulation. J. Mol. Biol. 1996;257:430–440. doi: 10.1006/jmbi.1996.0173. [DOI] [PubMed] [Google Scholar]
  • 6.Li A.J., Daggett V. Identification and Characterization of the Unfolding Transition-State of Chymotrypsin Inhibitor 2 by Molecular-Dynamics Simulations. J. Mol. Biol. 1996;257:412–429. doi: 10.1006/jmbi.1996.0172. [DOI] [PubMed] [Google Scholar]
  • 7.Ladurner A.G., Itzhaki L.S., Daggett V., Fersht A.R. Synergy between Simulation and Experiment in Describing the Energy Landscape of Protein Folding. Proc. Natl. Acad. Sci. U.S.A. 1998;95:8473–8478. doi: 10.1073/pnas.95.15.8473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Daggett V., Li A.J., Fersht A.R. Combined Molecular Dynamics and Phi-Value Analysis of Structure-Rreactivity Relationships in the Transition State and Unfolding Pathway of Barnase: Structural Basis of Hammond and Anti-Hammond Effects. J. Amer. Chem. Soc. 1998;120:12740–12754. [Google Scholar]
  • 9.Egan D.A., Logan T.M., Liang H., Matayoshi E., Fesik S.W., Holzman T.F. Equilibrium Denaturation of Recombinant Human FK506 Binding Protein in Urea. Biochemistry. 1993;32:1920–1927. doi: 10.1021/bi00059a006. [DOI] [PubMed] [Google Scholar]
  • 10.Logan T., Theriault Y., Fesik S. Structural Characterization of the FK506 Binding-Protein Unfolded in Urea and Guanidine-Hydrochloride. J. Mol. Biol. 1993;236:637–648. doi: 10.1006/jmbi.1994.1173. [DOI] [PubMed] [Google Scholar]
  • 11.van Duyne G.D., Standaert R.F., Karplus P.A., Schreiber S.L., Clardy J. Atomic-Structure of FKBP-FK506, An Immunophilin-Immunosuppressant Complex. Science. 1991;252:839–842. doi: 10.1126/science.1709302. [DOI] [PubMed] [Google Scholar]
  • 12.Michnick S.W., Rosen M.K., Wandless T.J., Karplus M., Schreiber S.L. Solution Structure of FKBP, a Rotamase Enzyme and Receptor for FK506 and Rapamycin. Science. 1991;252:836–842. doi: 10.1126/science.1709301. [DOI] [PubMed] [Google Scholar]
  • 13.Jackson S.E., Fersht A.R. Folding of Chymotrypsin Inhibitor-2. 1. Evidence for a Two-State Transition. Biochemistry. 1991;30:10428–10435. doi: 10.1021/bi00107a010. [DOI] [PubMed] [Google Scholar]
  • 14.Fersht A.R. Optimization of Rates of Protein Folding: The Nucleation-Collapse Mechanism for the Folding of Chymotrypsin Inhibitor 2 (CI2) and Its Consequences. Proc. Natl. Acad. Sci. USA. 1995;92:10869–10873. doi: 10.1073/pnas.92.24.10869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Jackson S.E. How Do Small Single-Domain Proteins Fold? Folding & Design. 1998;3:R81–R90. doi: 10.1016/S1359-0278(98)00033-9. [DOI] [PubMed] [Google Scholar]
  • 16.Munoz V., Eaton W.A. A Simple Model for Calculating the Kinetics of Protein Folding from Three-Dimensional Structures. Proc. Natl. Acad. Sci. USA. 1999;96:11311–11316. doi: 10.1073/pnas.96.20.11311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Main E.R.G., Fulton K.F., Jackson S.E. Folding of FKBP12: Pathway of Folding and Characterisation of the Transition State. J. Mol. Biol. 1999;291:429–444. doi: 10.1006/jmbi.1999.2941. [DOI] [PubMed] [Google Scholar]
  • 18.Khorasanizadeh S., Peters I.D., Roder H. Evidence for a Three-State Model of Protein Folding from Kinetic Analysis of Ubiquitin Variants with Altered Core Residues. Nature Struct. Biol. 1996;3:193–205. doi: 10.1038/nsb0296-193. [DOI] [PubMed] [Google Scholar]
  • 19.Chen B., Baase W.A., Schellman J.A. Low Temperature Unfolding of a Mutant of Phage T4 Lysozyme. 2. Kinetic investigations. Biochemistry. 1989;26:691–699. doi: 10.1021/bi00428a042. [DOI] [PubMed] [Google Scholar]
  • 20.Fulton K.F., Main E.R.G., Daggett V., Jackson S.E. Mapping the Interactions Present in the Transition State for Folding/Unfolding of FKBP12. J. Mol. Biol. 1999;291:445–461. doi: 10.1006/jmbi.1999.2942. [DOI] [PubMed] [Google Scholar]
  • 21.Jackson S.E., el Masry N., Fersht A.R. Structure of the Hydrophobic Core in the Transition State for Folding of Chymotrypsin Inhibitor 2: A Critical Test of the Protein Engineering Method of Analysis. Biochemistry. 1993;32:11270–11278. doi: 10.1021/bi00093a002. [DOI] [PubMed] [Google Scholar]
  • 22.Main E.R.G., Fulton K.F., Jackson S.E. The Context-Dependent Nature of Destabilising Mutations on The Stability of FKBP12. Biochemistry. 1998;37:6145–6153. doi: 10.1021/bi973111s. [DOI] [PubMed] [Google Scholar]
  • 23.Li A.J., Daggett V. Characterization Of The Transition-State Of Protein Unfolding By Use Of Molecular-Dynamics-Chymotrypsin Inhibitor-2. Proc. Natl. Acad. Sci. U.S.A. 1994;91:10430–10434. doi: 10.1073/pnas.91.22.10430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Main E.R.G., Jackson S.E. Does Trifluoroethanol Affect Folding Pathways and Can It be Used as a Probe of Structure in Transition States? Nature Struct. Biol. 1999;6:831–835. doi: 10.1038/12287. [DOI] [PubMed] [Google Scholar]
  • 25.Kentsis A., Sosnick T.R. Trifluoroethanol Promotes Helix Formation by Destabilizing Backbone Exposure: Desolvation Rather than Native Hydrogen Bonding Defines the Kinetic Pathway of Dimeric Coiled Coil Folding. Biochemistry. 1998;37:14613–14622. doi: 10.1021/bi981641y. [DOI] [PubMed] [Google Scholar]
  • 26.Ionescu R.M., Matthews C.R. Folding under the Influence. Nature Struct. Biol. 1999;6:304–307. doi: 10.1038/7534. [DOI] [PubMed] [Google Scholar]
  • 27.Walgers R., Lee T.C., CammersGoodwin A. An Indirect Chaotropic Mechanism for the Sstabilization of Helix Cconformation of Peptides in Aqueous Trifluoroethanol and Hexafluoro-2-Propanol. J. Amer. Chem. Soc. 1998;120:5073–5079. [Google Scholar]
  • 28.Storrs R.W., Truckses D., Wemmer D.E. Helix Propagation in Trifluoroethanol Solutions. Biopolymers. 1992;32:1695–1702. doi: 10.1002/bip.360321211. [DOI] [PubMed] [Google Scholar]
  • 29.Matouschek A., Kellis J.T., Jr., Serrano L., Fersht A.R. Mapping the Transition State and Pathway of Protein Folding by Protein Engineering. Nature. 1989;342:122–126. doi: 10.1038/340122a0. [DOI] [PubMed] [Google Scholar]
  • 30.Matouschek A., Kellis J.T., Jr., Serrano L., Bycroft M., Fersht A.R. Transient Folding Intermediates Characterized by Protein Engineering. Nature. 1990;346:440–445. doi: 10.1038/346440a0. [DOI] [PubMed] [Google Scholar]
  • 31.Serrano L., Matouschek A., Fersht A.R. The Folding of an Enzyme. 3. Structure of the Transition State for Unfolding of Barnase Analysed by a Protein Engineering Procedure. J. Mol. Biol. 1992;224:805–818. doi: 10.1016/0022-2836(92)90563-y. [DOI] [PubMed] [Google Scholar]
  • 32.Itzhaki L.S., Otzen D.E., Fersht A.R. The Structure of the Transition State for Folding of Chymotrypsin Inhibitor 2 Analysed by Protein Engineering Methods: Evidence for a Nucleation-Collapse Mechanism for Protein Folding. J. Mol. Biol. 1995;254:260–288. doi: 10.1006/jmbi.1995.0616. [DOI] [PubMed] [Google Scholar]
  • 33.Viguera A.R., Serrano L., Wilmanns M. Different Folding Transition-States May Result In The Same Native Structure. Nat. Struct. Biol. 1996;3:874–880. doi: 10.1038/nsb1096-874. [DOI] [PubMed] [Google Scholar]
  • 34.Grantcharova V.P., Riddle D.S., Santiago J.V., Baker D. Important Role of Hydrogen Bonds in the Structurally Polarized Transition State for Folding of the src SH3 Domain. Nat. Struct. Biol. 1998;5:714–720. doi: 10.1038/1412. [DOI] [PubMed] [Google Scholar]
  • 35.Lopez-Hernandez E., Serrano L. Structure of the Transition State for Folding of the 129 aa Protein CheY Resembles that of a Smaller Protein, CI-2. Folding & Design. 1996;1:43–55. [PubMed] [Google Scholar]
  • 36.Villegas V., Martinez J.C., Aviles F.X., Serrano L. Structure of the Transition State in the Folding Process of Human Procarboxypeptidase A2 Activation Domain. J. Mol. Biol. 1998;283:1027–1036. doi: 10.1006/jmbi.1998.2158. [DOI] [PubMed] [Google Scholar]
  • 37.Matthews J.M., Fersht A.R. Exploring the Energy Surface of Protein Folding by Structure-Reactivity Relationships and Engineered proteins: Observation of Hammond Behaviour for the Gross Structure of the Transition State and Anti-Hammond Behaviour for Structural Elements for Unfolding/Folding of Barnase. Biochemistry. 1995;34:6805–6814. doi: 10.1021/bi00020a027. [DOI] [PubMed] [Google Scholar]
  • 38.Miranker A., Radford S.E., Karplus M., Dobson C.M. Demonstration by NMR of Folding Domains in Lysozyme. Nature. 1991;349:633–636. doi: 10.1038/349633a0. [DOI] [PubMed] [Google Scholar]
  • 39.Radford S.E., Dobson C.M., Evans P.A. The Folding of Hen Lysozyme Involves Partially Structured Intermediates and Multiple Pathways. Nature. 1992;358:302–307. doi: 10.1038/358302a0. [DOI] [PubMed] [Google Scholar]
  • 40.Callihan D., Logan T.M. Conformations of Peptide Fragments from the FK506 Binding Protein: Comparison with the Native and Urea-Unfolded States. J. Mol. Biol. 1999;285:2161–2175. doi: 10.1006/jmbi.1998.2440. [DOI] [PubMed] [Google Scholar]
  • 41.Fersht A.R. Nucleation Mechanisms in Protein Folding. Curr. Opin. Struct. Biol. 1997;7:3–9. doi: 10.1016/s0959-440x(97)80002-4. [DOI] [PubMed] [Google Scholar]
  • 42.Wetlaufer D.B. Nucleation, Rapid Folding, and Globular Intrachain Regions in Proteins. Proc. Natl. Acad. Sci. U.S.A. 1973;70:697–701. doi: 10.1073/pnas.70.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wetlaufer D.B. Nucleation in Protein Folding-Confusion of Structure and Process. Trends Biochem. Sci. 1990;15:414–415. doi: 10.1016/0968-0004(90)90275-g. [DOI] [PubMed] [Google Scholar]
  • 44.Wang A., Bolen D.W. A Naturally Occurring Protective System in Urea-Rich Cells: Mechanism of Osmolyte Protection of Proteins Against Urea Denaturation. Biochemistry. 1997;36:9101–9108. doi: 10.1021/bi970247h. [DOI] [PubMed] [Google Scholar]
  • 45.Kraulis P. Molscript, a Program to Produce Both Detailed and Schematic Plots of Protein Structures. J. Appl. Crystallogr. 1991;24:946–950. [Google Scholar]
  • 46.Merritt E.A., Murphy M.E.P. Raster3D Version-2.0-A Program For Photorealistic Molecular Graphics. Acta Crystallogr. D. 1994;50:869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]

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