Analysis of Metal Effects on C‐Peptide Structure and Internalization

Abstract

The connecting peptide (C‐peptide) has received increased attention for its potential therapeutic effects in ameliorating illnesses such as kidney disease and diabetes. Although the mechanism of C‐peptide signaling remains elusive, evidence supports its internalization and intracellular function. Emerging research is uncovering the diverse biological roles metals play in controlling and affecting the function of bioactive peptides. The work presented herein investigates interactions between C‐peptide and first‐row d‐block transition metals, as well as their effects on C‐peptide internalization into cells. Through spectroscopic techniques, it is demonstrated that CrIII, CuII, and ZnII bind to C‐peptide with differing stoichiometries and biologically relevant affinities. In addition, metal binding elicits both subtle changes in secondary structure and inhibits adoption of an α‐helical character in environments where the dielectric constants are reduced. This study shows how metal ions can modulate peptide hormone activity through subtle structural changes to disrupt cellular uptake.

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity

“…The formation constant determined here for C-peptide is consistent with what we previously reported. 32 The use of ITC allows for the decomposition of the free energy into its enthalpic and entropic contributions, and from this inspection, we see that Cu(II) binding is predominantly entropically driven. This favorable entropy may be the result of the desolvation of both C-peptide at the metal-binding site and the Cu(II) ion (the latter of which can have up to 10 water molecules surrounding it), 50 as well as the chelate effect.…”

“…On the basis of electronic absorption spectroscopy studies, we previously hypothesized that Cu(II) could be binding to a combination of the backbone amide nitrogens and the carboxylate side chains for a mixed N x O y ligand set. 32 To assess this hypothesis, we turned to electron paramagnetic resonance (EPR) spectroscopy, which probes the ligand environment around the paramagnetic Cu(II) (3d 9 , S = 1/2) center. For instance, if a nitrogen is directly bound to Cu(II), then the 14 N-hyperfine coupling is strong enough (∼45 MHz) to yield characteristic hyperfine splittings in the X-band EPR spectrum, although the absence of hyperfine splitting does not preclude direct nitrogen coordination as evidenced by previous studies performed with Cu(II)/amyloid-β complexes.…”

“…In our previous work, we calculated an apparent binding constant of Cu(II) to C-peptide in the 15−40 nM range via competition with the chromophoric ligand, phenanthroline. 32 Using this range as a starting point, Cu(II) was titrated into C-peptide in MOPS and MOPSO buffers, which were chosen for their small K Cu(II)-buffer (Figure 8 and Figure S7), 47 in triplicate, and the experimental fit parameters are listed in Table S6. (There is a dearth of buffers with small K Cu(II)-buffer that will allow for the study of low-affinity peptides and proteins.)…”

“…Additionally, we determined that, in solution, Cu(II) directly interacts with C-peptide. 32 T h i s c o n t e n t i s Cu(II) binding was an intriguing discovery given that the peptide sequence does not contain classical copper binding residues, potentially pointing to the anchoring of Cu(II) through the terminal amine as seen with other peptides. Precisely defining this binding site poses challenges that are inherent to the structural aspects of the peptide.…”

“…In addition, our earlier work did not show any metal-specific alterations in the overall structure that could otherwise be used to probe the interaction. 32 Second, the peptide sequence (EAEDLQVGQVELGGGPGAGSLQPL-ALEGSLQ) is low in complexity, bearing only nine different residues, with glycine appearing seven times. Elucidating the metal binding site thus requires the question to be approached via multiple techniques to dissect the various pieces that contribute to the interaction.…”