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Two exposed amino acid residues confer thermostability on a cold shock protein


Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol−1 at 70 °C). 11.5 kJ mol−1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

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Figure 1: The mesophilic cold shock protein Bs-CspB differs from its thermophilic homolog, Bc-Csp, at 12 sequence positions.
Figure 2: Thermal stability curves.
Figure 3: Effect of all individual sequence differences on the stability of Bc-Csp.
Figure 4: The residues that contribute most to the difference in thermal stability between Bs-CspB and Bc-Csp are located near each other at the protein surface.


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We thank the members of our laboratories and the Marahiel laboratory for help and discussions as well as C. Brooks III (Scripps Research Institute) and C. Nick Pace (Texas A&M University) for a fruitful exchange of ideas about the electrostatic stabilization of proteins. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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Correspondence to Franz X. Schmid.

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Perl, D., Mueller, U., Heinemann, U. et al. Two exposed amino acid residues confer thermostability on a cold shock protein. Nat Struct Mol Biol 7, 380–383 (2000).

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