The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability.
The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability has been quantitated using de novo designed synthetic coiled-coils. The synthesized model coiled-coil (EK), denoted by amino acid residues in positions e and g, which contains only interhelical ionic interactions without any possible (i, i + 3) and (i, i + 4) intrahelical ionic interaction, consists of two identical 35 residue polypeptide chains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coils were prepared where five Glu residues at e positions in EK were mutated to Gln residues (QK); five Lys residues at g positions were altered to Gln residues (EQ) or these mutations were effected at both positions e and g (QQ). The stabilities of the four coiled-coils were determined by measuring the ellipticities at 220 nm as a function of urea concentration at 20 degrees C. By using a double-mutant cycle analysis it was possible to isolate the energetic contribution of interhelical ionic attractions to coiled-coil stability from the other contributions such as helical preference and hydrophobicity. The 0.37 +/- 0.01 kcal/mol of energetic contribution of one interhelical ion pair to the coiled-coil stability was obtained from three independent comparisons. This findings suggests that a large number of weak interhelical electrostatic interactions on the surface of a protein can make a substantial contribution to protein stability. In addition, the energetic contributions of a single mutation E(-)-->Q, K(+)-->Q, Q-->E degrees and E(-)-->E degrees were also determined (delta delta G = 0.22, 0.26, and 0.46 and 0.65 kcal/mol for the single mutations, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Zhou
N E
NE
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
Hodges
R S
RS
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D007477:Ions; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014508:Urea / Q000494:pharmacology
D007477:Ions; D010455:Peptides; D014508:Urea