An Engineered β‐Hairpin Peptide Forming Thermostable Complexes with Zn^II, Ni^II, and Cu^II through a His₃ Site

Abstract

The three-dimensional structure of a peptide, which determines its function, can denature at elevated temperatures, in the presence of chaotropic reagents, or in organic solvents. These factors limit the applicability of peptides. Herein, we present an engineered β-hairpin peptide containing a His 3 site that forms complexes with Zn II , Ni II , and Cu II . Circular dichroism spectroscopy shows that the peptideÀ metal complexes exhibit melting temperatures up to 80 °C and remain folded in 6 M guanidine hydrochloride as well as in organic solvents. Intrinsic fluorescence titration experiments were used to determine the dissociation constants of metal binding in the nano-to subnanomolar range. The coordination geometry of the peptideÀ Cu II complex was studied by EPR spectroscopy, and a distorted square planar coordination geometry with weak interactions to axial ligands was revealed. Due to their impressive stability, the presented peptideÀ metal complexes open up interesting fields of application, such as the development of a new class of peptideÀ metal catalysts for stereoselective organic synthesis or the directed design of extremophilic β-sheet peptides.

Design of Functional Globular β‐Sheet Miniproteins

“…Moreover, Tz2H 3 -Cu(II) was also folded in organic solvents such as methanol or a methanol-acetonitrile mixture, whereas apo-Tz2H 3 and the original Trpzip2 were only weakly folded. [60] Metal binding was also reported for a 16 amino acid β-hairpin with a His 2 Cys 2 site placed at the non-HB sites (Figure 4B). This peptide WHC bound to Cu(II) > Ni(II), Zn(II) (relative affinities by competition experiments, no K d values were reported).…”

“…Under such high concentrations of chaotropic salts, most proteins are completely denatured. Moreover, Tz2H 3 ‐Cu(II) was also folded in organic solvents such as methanol or a methanol‐acetonitrile mixture, whereas apo‐Tz2H 3 and the original Trpzip2 were only weakly folded [60] …”

Crystallography reveals metal-triggered restructuring of β-hairpins

“…Though no crystal structures had existed for any trpzip peptides prior to this work, NMR studies and analytical ultracentrifugation provided unambiguous structural evidence that previous trpzip derivatives form monomeric hairpin motifs. [7,11,12] The structure of Cu-TZ4H, however, is a domain-swapped dimer, wherein the expected hairpin turn has instead straightened, and two of these chains are brought together by two Cu II ions. In line with the observations by CD spectroscopy, the antiparallel zippered βsheet motif still appears in the domain-swapped dimer.…”

“…Though there are numerous crystal structures for ɑhelical metallopeptides, to the best of our knowledge, no crystal structures have ever been reported for canonical metallo-βsheets. [11][12][13][14] Note that there are examples of noncanonical, modified β-sheet-based molecules that have been crystallized with bound metal ions, but the fundamental architecture of these molecules differs significantly from a that of metalloproteins, and thus are not considered metallo-β-sheets. [15,16] The highest resolution structures for metallo-β-sheets have instead come from solid-state nuclear magnetic resonance (NMR) and cryo-electron microscopy, however the resolution is low (3.48 Å for the latter).…”

“…Though metalfree, soluble β-sheets are well-characterized by NMR spectroscopy and crystallography, [7,14,[19][20][21] their metal complexes have mainly been characterized by circular dichroism (CD). [11,12] Since CD only reports on secondary structure, [22] the tertiary and quaternary structure are poorly understood for metallo-β-sheets.…”