Contributions of folding cores to the thermostabilities of two ribonucleases H - PubMed (original) (raw)
Comparative Study
Contributions of folding cores to the thermostabilities of two ribonucleases H
Srebrenka Robic et al. Protein Sci. 2002 Feb.
Abstract
To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (DeltaCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low DeltaCp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.
Figures
Fig. 1.
Sequence alignment of T. thermophilus and E. coli RNases H* (cysteine-free variants of RNase H). Vertical lines separate the core region from the remaining part (outside) of the protein. The bold letters indicate the sequence of TCEO chimera (T. thermophilus Core E. coli Outside), whereas the non-bold letters correspond to the sequence of ECTO chimera (E. coli Core T. thermophilus Outside). A cartoon of secondary structural elements (rectangles labeled αA–αE correspond to helices; arrows labeled β1–β5 correspond to strands) is shown below the sequences.
Fig. 2.
Crystal structures of (left) E. coli RNase H* (Goedken et al. 2000) and (right) T.thermophilus RNase H (Ishikawa et al. 1993). Folding cores are shown in dark grey.
Fig. 3.
CD spectra of E. coli RNase H* (□) T. thermophilus RNase H* (○), ECTO (▾) and TCEO(▴) chimera.
Fig. 4.
Ribbon diagram of the crystal structure of TCEO (shown in black) overlaid with the structure of E. coli RNase H* (shown in light gray) (Goedken et al. 2000).
Fig. 5.
Thermodynamic stability of TCEO (▴) and ECTO (▾) compared with E. coli (□) and T. thermophilus RNases H* (○): (a) Thermal melts in 1 M guanidium chloride (E. coli RNase H thermal denaturation is not reversible in the absence of denaturants); (b) Guanidinium chloride melts, (c) Urea melts. The error bars correspond to errors of fits to a two-state model.
Fig. 5.
Thermodynamic stability of TCEO (▴) and ECTO (▾) compared with E. coli (□) and T. thermophilus RNases H* (○): (a) Thermal melts in 1 M guanidium chloride (E. coli RNase H thermal denaturation is not reversible in the absence of denaturants); (b) Guanidinium chloride melts, (c) Urea melts. The error bars correspond to errors of fits to a two-state model.
Fig. 5.
Thermodynamic stability of TCEO (▴) and ECTO (▾) compared with E. coli (□) and T. thermophilus RNases H* (○): (a) Thermal melts in 1 M guanidium chloride (E. coli RNase H thermal denaturation is not reversible in the absence of denaturants); (b) Guanidinium chloride melts, (c) Urea melts. The error bars correspond to errors of fits to a two-state model.
Fig. 6.
Stability curves TCEO (• and ▪) and ECTO (○ and □) compared with the stability curves of T. thermophilus (broken line) and E. coli RNases H (dotted line) (Hollien and Marqusee 1999b). Each circle corresponds to a ΔG obtained from a guanidine denaturation experiment. Squares correspond to ΔGs obtained from temperature denaturation experiments. The dash-dot plot represents the constrained fit of TCEO stability data to the Gibbs-Helmholtz equation, in which ΔCp is fixed at 2.2 kcal mole−1 K−1. Fitting the stability curves to Gibbs-Helmholtz equation (without any constraints) results in ΔCp of 1.6 ± 0.2 kcal mole–1 K−1, Tm of 76 ± 1°C, and ΔH of 68 ± 2 kcal for TCEO; and ΔCp of 2.4 ± 0.3 kcal mole−1 K−1, Tm of 61 ± 1°C, and ΔH of 89 ± 2 kcal for TCEO.
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References
- Bailey, S. 1994. The Ccp4 suite – programs for protein crystallography. Acta Crystallogr. Sect. D-Biol. Crystallogr. 50 760–763. - PubMed
- Becktel, W.J. and Schellman, J.A. 1987. Protein stability curves. Biopolymers 26 1859–1877. - PubMed
- Black, C.B. and Cowan, J.A. 1994. Magnesium aactivation of ribonuclease H – evidence for one catalytic metal ion. Inorganic Chem. 33 5805–5808.
- Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. Sect. D Biol.l Crystallogr. 54 905–921. - PubMed
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