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.