Functional, metal-based crosslinkers for α-helix induction in short peptides

Abstract

Many protein-protein interactions that play a central role in cellular processes involve α-helical domains. Consequently, there has been great interest in developing strategies for stabilizing short peptides in α-helical conformations toward the inhibition and interrogation of protein-protein interactions. Here, we show that tridentate Hybrid Coordination Motifs (HCMs), which consist of a natural (histidine, His) and an unnatural (8-hydroxyquinoline, Quin) metal binding functionality, can bind divalent metal ions with high affinity and thereby induce/stabilize an α-helical configuration in short peptide sequences. The Quin functionality is readily introduced onto peptide platforms both during or after solid-state peptide synthesis, demonstrating the preparative versatility of HCMs. A systematic study involving a series of HCM-bearing peptides has revealed the critical importance of the length of the linkage between the Quin moiety and the peptide backbone as well as the metal coordination geometry in determining the extent of α-helix induction. Through Zn^II coordination or modification with Re^I(Quin)(CO)₃, the HCM-bearing peptides can be rendered luminescent in the visible region, thus showing that HCMs can be exploited to simultaneously introduce structure and functionality into short peptides.

Figure 1

(a) Proposed scheme for α-helix induction through tridentate metal coordination by an HCM. The remaining coordination sites on the metal are likely filled by aquo ligands. (b) Chemical structures of various Quin functionalities. (c) Sequences of peptide constructs prepared in this study. The coloring scheme corresponds to that in (a).

Figure 2

Changes in the circular dichroism spectra of HCM-bearing peptides upon binding Cu^II and Zn^II. Spectra were acquired at 4 °C with 10 μM peptide and 30 μM EDTA or metal in 5 mM sodium borate buffer at pH 7.5. Corresponding spectra for Co^II and Ni^II are shown in Figure S9 and S10.

Figure 3

(a) H-EMeQuin fluorescence upon metal addition (λ_exc = 385 nm). (b) Normalized fluorescence intensity (F/F₀) of each peptide when bound to Zn^II as compared to the metal-free peptide.

Figure 4

(a) Proposed scheme for the formation of the Re(Quin)(CO)₃(His) HCM upon heating and the subsequent formation of the α-helix upon cooling. (b) Observed changes in the CD spectrum upon the heating and subsequent cooling of the Re-HCM peptide. (c) Changes in the CD signal (222 nm) during heating and cooling. (d) The emission spectrum of the Re-HCM peptide obtained after the heating/cooling cycle (λ_exc = 410 nm). (e) The IR spectrum of the Re-HCM peptide. The peptide sample was first lyophilized and then combined with KBr to make a pellet.

Figure 5

Sequences of wild-type and HCM-modified Bax BH3 domain peptides (top). Far-UV CD spectra of the Bax-HCM in the presence and absence of Cu^II (blue) and Zn^II (magenta) acquired at 4 °C (solid lines) or 25 °C (dashed lines). The samples contained 10 μM peptide and 30 μM EDTA or metal ion in a 5 mM sodium borate buffer solution at pH 7.5.