Selective Formation of AAB‐ and ABC‐Type Heterotrimeric α‐Helical Coiled Coils

Abstract

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.

Optimization of aromatic side chain size complementarity in the hydrophobic core of a designed coiled‐coil

“…This design has been further expanded at the f ‐position, in order to increase the specificity of the AAB‐type coiled‐coil interaction. Then, IZ‐2A( f A) and IZ‐2W( f Q), where the f ‐positions were occupied by Ala residues and Gln residues, respectively, were shown to selectively form the [IZ‐2A( f A)] 2 /IZ‐2W( f Q) complex (25). IZ‐2A( f A) and IZ‐2W( f Q) only exhibited random coil structure, whereas their 2 : 1 mixture formed the [IZ‐2A( f A)] 2 /IZ‐2W( f Q) complex.…”

“…Although IZ‐2F( f Q) formed the trimeric structure by itself, the selective formation of an AAB‐type heterotrimer would also be possible, with further design. For example, it could be accomplished by selecting the amino acid residues at the f ‐position to fine‐tune the thermal stability (25).…”

“…Because the improved packing of the protein structure could lead to an increase in the thermal stability (21,22), the design of the packing structure of the protein interior is an important de novo target. Successful designs for the control of the orientation and the selective assembly of peptide fragments of coiled‐coils have been reported (23–31).…”

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“…6,7 The fluorescence of this compound allows for convenient quantification and potential use as labeling agents for any future in vivo applications. 3-Carboxycoumarins can be conveniently and efficiently synthesized at room temperature in water 8 from 2-hydroxybenzaldehydes and…”