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

Thermophilic organisms produce proteins of exceptional stability. To unders tand protein thermostability at the molecular level we studied a pair of co ld shock proteins, one of mesophilic and one of thermophilic origin, by sys tematic mutagenesis, Although the two proteins differ in sequence at 12 pos itions, two surface-exposed residues are responsible for the increase in st ability of the thermophilic protein (by 15.8 kJ mol(-1) at 70 degrees C). 1 1.5 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 ca rboxy terminus. The mesophilic protein could be converted to a highly therm ostable 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 sim ple and powerful approach for increasing the thermostability of a protein.