Direct evidence for alteration of unfolding profile of a helical peptide by far‐ultraviolet circular dichroism aromatic side‐chain contribution

Abstract

Aromatic side-chains are known to contribute to the far-UV circular dichroism (CD) spectra of peptides and proteins. Among other things, this can signi¢cantly a¡ect the measured helix propensities of amino acids [Chakrabartty et al., Biochemistry 32 (1993) 5560^5565]. In order to address how interfering side-chain contributions can a¡ect the backbone unfolding transition of a helical peptide, as monitored by [a a] 222 (molar ellipticity at 222 nm), we have studied the unfolding transition of a short designed (K K-amino isobutyric acid/alanine-based) helical peptide containing an interacting Tyr residue. The guanidinium hydrochloride-induced unfolding of the peptide, as monitored by [a a] 222 , showed the presence of a sharp transition superposed over a much broader transition. When the same experiment was performed with a similar peptide that lacked the interacting Tyr residue, the sharp transition disappeared and only the broad transition remained. The sharp transition was assigned to originate from the interacting Tyr sidechain. This demonstrates that conformationally restricted aromatic side-chains that interact with the helical backbone not only can alter the backbone far-UV CD signal, they may also alter the unfolding pro¢les, monitored by far-UV CD, rendering them un¢t for a simple analysis for extracting the appropriate unfolding thermodynamic parameters. ß

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

“…Typically Tyr is introduced in a synthetic peptide for accurate concentration measurement. MD results showed that Tyr side chain could affect the inherent helical propensities of the designed backbone, confirming our earlier experimental study 28…”

“…The synthesis, purification and characterization of the peptides studied, ABK (Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH 2 ) and ABGY (Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Gly‐Gly‐Tyr‐NH 2 ), have been reported earlier 28, 29. The peptides contain 42% Ala with a strong preference for the α‐helical backbone, 25% Aib (single letter code: B) with a strong preference for the 3 10 ‐helix making them ideal candidates for exhibiting the α/3 10 ‐helix transition.…”

“…Lys residues were suitably placed for water solubility and prevention of aggregation due to repulsion of positive side‐chains at neutral or low pH. The sequences of ABK and ABGY were identical except for a (Gly) 2 spacer in ABGY to rule out interference of Tyr side‐chain contribution in CD experiments 28. NMR experimental data reported were performed on ABK to avoid resonance overlap found in ABGY, whereas CD experimental data reported were performed with ABGY.…”

“…As part of ongoing work in our laboratory, we recently designed two water‐soluble (designed by suitably placed Lys residues) helical (induced by Ala/Aib) peptides, ABK and ABGY 28, 29. From unfolding experiments, induced by urea and guanidium hydrochloride, helical conformations adopted by these peptides were shown to be as stable as an Ala‐based peptide helix twice its length 29.…”

Type VIa β‐turn‐fused helix N‐termini: A novel helix N‐cap motif containing cis proline

“…The shapes of the CD spectra of the peptides (Supporting Information Fig. S3), K7 (Ac‐A EPYAS BKBAKA‐NH 2 ) and K8 (Ac‐A EPYAS BAKBKAA‐NH 2 ), were similar to the CD spectra of ABK (Ac‐ABAKABKAKABY‐NH 2 ) and ABGY (Ac‐ABAKABKAKABGGY‐NH 2 ), two helical peptides containing Ala/Aib of similar length . However, the CD band intensities of K7 and K8 were reduced by ∼50%, not unexpected because of the higher Ala/Aib content in ABK/ABGY and the presence of Pro in K7 and K8.…”