Destabilization of a protein helix by electrostatic interactions.
Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.
Walter
S
S
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Hubner
B
B
Hahn
U
U
Schmid
F X
FX
D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D017931:DNA Primers / Q000737:chemistry; D005971:Glutamates / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011994:Recombinant Proteins; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000648:ultrastructure; D013816:Thermodynamics
D017931:DNA Primers; D005971:Glutamates; D007477:Ions; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D006163:Ribonuclease T1