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Design and synthesis of the pseudo‐EF hand in calbindin D 9K : Effect of amino acid substitutions in the α‐helical regions
Author(s) -
Tsuji Takashi,
Kaiser Emil T.
Publication year - 1991
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340090103
Subject(s) - crystallography , helix (gastropod) , chemistry , salt bridge , amino acid , side chain , peptide , stereochemistry , peptide sequence , calcium , alpha helix , mutant , biochemistry , circular dichroism , biology , ecology , organic chemistry , snail , gene , polymer
A series of 37‐residue analogues of the pseudo‐EF hand in bovine calbindin D 9K has been synthesized by the solid phase method. In the presence of calcium an α‐helical induction of up to 44% was observed for the peptide with the native sequence with a K d for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two α‐helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu − …Lys + salt bridge or by changing the N‐terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices. The mutants with the substitution of (Leu‐30, Leu‐32) to (Gln‐30, Gln‐32), (Gln‐30, Ala‐32), and (Ala‐30, Ala‐32) designed based on the strategy 1 do not show any affinity for calcium and have low α‐helicity. The Leu‐30 to Lys‐30 mutant designed to form a salt bridge between the side chains of Glu‐26 and Lys‐30 has an apparent K d for calcium of 6.8 mM. K d of the N‐terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the α‐helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr‐13, Leu‐31 to Cys‐31 and Tyr‐13, Leu‐31 to Cys‐13, hCys‐31, show apparent K d values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest α‐helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix–loop–helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.