Journal of Molecular Biology
Regular articleA comparison of the folding kinetics and thermodynamics of two homologous fibronectin type III modules1
Introduction
The spontaneous refolding of proteins occurs with time constants ranging from milliseconds to hours (e.g. see Jackson and Fersht 1991, Huang and Oas 1995, Kragelund et al 1995, Kern et al 1995). While primary sequence is presumably the ultimate determinant of these rates, many factors such as protein size, topology, proline content and thermodynamic stability are thought to play a role in this large range of kinetic behaviour Creighton 1978, Kiefhaber et al 1992, Orengo et al 1994, Bryngelson et al 1995, Fersht 1995, Onuchic et al 1995, Gross 1996, Mines et al 1996, Miranker and Dobson 1996. Discriminating between the contributions of each of these properties to the kinetics of protein folding would require the ability to vary each characteristic independently. While this has not yet proved feasible, the study of the refolding kinetics of members of homologous protein families should provide a means of examining the role that sequence and stability play in determining folding kinetics in the absence of complicating factors such as significantly different topologies or structures.
One of the largest known families of homologous proteins is that of the fibronectin type III modules (FNIII), of which more than 400 examples are known (Bork et al., 1996). In order to investigate the relationship between the folding characteristics of distantly homologous proteins, we have investigated the folding thermodynamics and kinetics of two members of this family, the ninth and tenth FNIII modules of human fibronectin (9FNIII and 10FNIII). These modules comprise seven β-strands arrayed in two topologically complex sheets (Figure 1: Main et al., 1992). The 10FNIII module contains eight proline residues, with at least one in each of the seven intra-strand loops. Previous research indicates that despite its proline-rich all β-sheet structure the 10FNIII module refolds very rapidly; in 0.64 M guanidine hydrochloride (GuHCl) the domain appears to recover fully native core and backbone structures within one second at 5°C (Plaxco et al., 1996). The 9FNIII module is slightly smaller (90 residues instead of 94) and contains only seven proline residues. We report here a detailed thermodynamic and kinetic analysis of the folding of both the 10FNIII and 9FNIII domains. The folding kinetics and thermodynamics of a related FNIII module, the third FNIII module of tenascin (TnfN3) are described by Clarke et al. (1997) in the accompanying paper.
Section snippets
Equilibrium stability
Equilibrium chemical denaturation studies indicate that the 10FNIII module is significantly more stable than its homologue (Figure 2). With an equilibrium stability (ΔGf) of −6.1(±0.1) kcal mol−1, the 10FNIII module is relatively stable for such a small, non-disulphide-bonded protein. Conversely, equilibrium denaturation studies suggest the 9FNIII module is relatively unstable, with a ΔGf of only −1.2(±0.5) kcal mol−1. Unfortunately, due to the relatively low stability of the module, it has not
Discussion
Previously reported studies suggest that structural differences between these two homologous domains are small despite a relatively low level of sequence identity (Leahy et al., 1996). Sequence alignment of 9FNIII and 10FNIII indicates that the two modules share 28% sequence identity (Figure 1: Kornblihtt et al., 1985). Sequence and structural alignments both show that 10FNIII contains a four-residue insertion lacking in 9FNIII. This element, comprised of residues 78 to 81 of 10FNIII,
Protein purification and equilibrium denaturation
Recombinant human 9FNIII and 10FNIII were expressed as individual proteins in the form of glutathione-S-transferase fusions in Escherichia. coli and purified as described (Mardon & Grant, 1994). Equilibrium unfolding experiments were conducted on samples incubated with various concentrations of GuHCl in 20 mM sodium acetate (pH 5.2) for ten minutes with a Perkin Elmer LS 50B fluorimeter held at to 25(±1)°C. Excitation was at 280 nm, and emission was monitored at 350(±10) nm. Final GuHCl
Acknowledgements
We thank Jane Clarke for many valuable discussions and for sharing unpublished data, Helen Mardon and Kate Grant for providing the FNIII-GST fusion clones, Sophie Jackson for providing PPI, Cameron Marshall for aid in conducting equilibrium unfolding experiments, Craig Morton for providing artistic advice, and Jay Winkler, Don Haynie and Nick Woodruff for communicating unpublished results. The Oxford Centre for Molecular Sciences is supported by the U.K. Biotechnology and Biological Sciences
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Edited by P. E. Wright
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Present addresses: K. W. Plaxco, Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195, USA; C. Spitzfaden, SmithKline Beecham Pharmaceuticals, Computational and Structural Sciences, Harlow, CM19 5AW, England.