Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10⁻¹⁵ M

Abstract

We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g ↔ eЈ (i, iЈ +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T m ‫ס‬ 28°C) or nanomolar (T m ‫ס‬ 40°C) range, respectively, but heterodimerize with a T m >90°C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 M and 6°C but that heterodimerize with a T m ‫ס‬ 74°C or K d(37) ‫ס‬ 1.1 × 10 −11 M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T m ‫ס‬ 73°C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

“…6B) indicate that E5/K5-coil-EGF remains biologically active following its immobilization on an E-or K-coil surface. Other groups have reported on the use of similar self-assembling peptide heterodimers based on human B-ZIP leucine zipper proteins for the generation of functionalized surfaces [49]. In this approach, one peptide was fused to a cysteine, which allowed for grafting to a polyacrylamide gel, whereas the other peptide was coupled to the RDGS cell adhesion binding domain.…”

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

“…In contrast to pIL, which contains an exclusively hydrophobic dimerization interface with probably three interhelical salt bridges (5, 9), pER contains a single asparagine residue at the a position and as many as eight interhelical salt bridges formed by E and R pairs (14). Whereas hydrophilic residues in the dimerization interface destabilize the coiled coil, the interhelical salt bridges enhance the affinity of the coiled coil (9).…”

“…To characterize the mechanisms of helix staggering and sliding and their roles in coiled coil folding and misfolding, we used highresolution optical tweezers to investigate two of the strongest coiled coils that have canonical sequence compositions: a variant of the GCN4 leucine zipper (pIL) (9), in which three valine residues and one asparagine residue in the a positions in the wild type have been replaced by the most stable isoleucine (13), and a heterodimer (pER) composed of oppositely charged glutamic acid and arginine residues at the e and g positions, respectively (14). Both parallel coiled coils have extraordinary stabilities, with melting temperatures above 100°C.…”

Reversible pH-controlled DNA-binding peptide nanotweezers: An in-silico study

“…Finally, in mutant M3CT we replaced the His-tag of mutant M3 with a pentaglycine tag. These mutation to the surface of cancer cells (Moll et al 2001). Coiled-coil proteins have been extensively investigated using various experimental techniques such as NMR (Nikolaev and Pervushin 2007), X-ray diffraction (O'Shea et al 1991), circular dichroism (CD) and fl uorescence spectroscopy (Suzuki et al 1998), differential scanning calorimetry (DSC) (Yu et al 1996) and electron spin resonance (ESR) spectroscopy (Columbus and Hubbell 2004) as well as theoretical and computational approaches using molecular dynamics (MD) simulation techniques (Mohanty et al 1999;Missimer et al 2005;Pineiro et al 2005).…”