Programmed Self-Sorting of Coiled Coils with Leucine and Hexafluoroleucine Cores

Abstract

Specific protein-protein interactions are crucial for virtually all biological processes. 1 Naturally occurring surfaces that mediate protein-protein interactions usually contain either elements of polar specificity, for example hydrogen bonding or salt bridges, or complementary hydrophobic patches that contain side chains that maximize van der Waals interactions. 2 We, 3 and others 4 have recently described a new type of protein-protein interaction interface that could potentially be both hydrophobic and lipophobic. This is accomplished by the introduction of nonproteinogenic, fluorine-containing amino acids. 5,6 Reported here is the design, synthesis, and programmed self-sorting of peptide systems with orthogonally miscible hydrocarbon and fluorous (highly fluorinated) cores.Peptides H and F are equipped with N-terminal cysteine residues and were designed to form parallel homodimeric coiled coil assemblies. 7 These peptides have an identical sequence except that all seven of the core leucine residues in H have been replaced by 5,5,5,5′,5′,5′-R-S-hexafluoroleucine in F (Figure 1), shielding 28 trifluoromethyl groups from aqueous solvent in the canonical fluorinated dimer. Hexafluoroleucine was synthesized according to a recently documented procedure. 8 The peptides were assembled on 4-methylbenzhydrylamine (MBHA) resin according to the in situ neutralization protocol for t-Boc peptide synthesis as described previously 9 and purified by reverse-phase HPLC. Purity of the peptides was confirmed by analytical HPLC and MALDI mass spectrometry. H and F are designed to form parallel coiled coil structures due to unfavorable interhelical electrostatic interactions in the antiparallel arrangements. 10 Furthermore, a single polar residue Asn14 was incorporated in the hydrophobic

Preferred side-chain constellations at antiparallel coiled-coil interfaces

“…The four ⌬G Urea values can be used to calculate DE LI (L/I) ϭ Ϫ3.2 kcal/mol for the Oakley system. In a complementary study, we prepared derivatives of the two acid and two base peptides bearing N-or C-terminal cysteine residues, respectively, to allow direct determination of the discrimination energy under native conditions by disulfide exchange (26)(27)(28). This approach indicated DE LI (L/I) ϭ Ϫ2.6 kcal/mol, in good agreement with the value deduced from the unlinked species.…”

Structural basis for the enhanced stability of highly fluorinated proteins

“…Thus, fluorous analogs of hydrophobic amino acids such as leucine, valine, and phenylalanine have been incorporated into both natural and de novo-designed proteins either biosynthetically or by chemical synthesis (18)(19)(20)(21). Proteins with sequences containing up to ∼25% fluorous residues have been synthesized without gross structural perturbation.…”

“…Proteins with sequences containing up to ∼25% fluorous residues have been synthesized without gross structural perturbation. In almost all cases, fluorination significantly enhances stability to thermal unfolding, chemical denaturation, and proteolytic degradation, with minimal impact on the biological activity of the protein or peptide (18,(21)(22)(23)(24).…”

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

“…In particular, we wanted to test whether preferential interactions between fluorinated residues, 15,16,34,35 sometimes referred to as the ''fluorous effect,'' were responsible for the stabilizing effects of fluorinated amino acids. This idea is predicated upon the unusual phase-segregating properties of perfluorocarbon solvents, which selectively extract highly fluorinated small molecules from organic solvents into the perfluorocarbon solvent.…”

“…[12][13][14] However, our laboratory and others have focused on introducing highly fluorinated analogs of residues such as valine, leucine, isoleucine, and phenylalanine into proteins as a means of enhancing stability. 12,[15][16][17][18][19][20][21][22][23] In most cases, these modified proteins display significantly increased stability toward unfolding by chemical denaturants and organic solvents, as well as increased resistance to proteolytic degradation. 20,[24][25][26] Currently, our understanding of how fluorination stabilizes protein folding is impeded by the lack of structures for proteins containing highly fluorinated amino acids.…”