Application of Alanine Scanning to Determination of Amino Acids Essential for Peptide Adsorption at the Solid/Solution Interface and Binding to the Receptor: Surface-Enhanced Raman/Infrared Spectroscopy versus Bioactivity Assays.

Edyta Proniewicz,Adam Prahl,Marta Makowska,Helena Domin,Grzegorz Burnat,Izabela Małuch

doi:10.1021/acs.jmedchem.1c00397

Abstract

The article describes the application of the alanine-scanning technique used in combination with Raman, surface-enhanced Raman, attenuated total reflection Fourier transform infrared, and surface-enhanced infrared absorption (SEIRA) spectroscopies, which allowed defining the role of individual amino acid residues in the C-terminal 6–14 fragment of the bombesin chain (BN6–14) on the path of its adsorption on the surface of Ag (AgNPs) and Au nanoparticles (AuNPs). A reliable analysis of the SEIRA spectra of these peptides was possible, thanks to a curve fitting of these spectra. By combining alanine-scanning with biological activity studies using cell lines overexpressing bombesin receptors and the intracellular inositol monophosphate assay, it was possible to determine which peptide side chains play a significant role in binding a peptide to membrane-bound G protein-coupled receptors (GPCRs). Based on the analysis of spectral profiles and bioactivity results, conclusions for the specific peptide–metal and peptide–GPCR interactions were drawn and compared.

Highlights

Vibrational spectroscopy [infrared absorption (IR) and Raman (RS)] is a widely used, reliable, and powerful method for studying conformational changes and molecular interactions and for unambiguously identifying and characterizing various molecules by their vibrational fingerprint
Our research has aimed to demonstrate that SERS and signal from conventional transmission or reflection experiments (SEIRA) can be used as potential tools to predict the biochemical activity of some neuropeptides.[6,18,19]
The side-chains of the aromatic amino acids have the greatest affinity for the metal surface; many of the observed bands can be assigned to normal vibrations based on the characteristic vibrations of the aromatic rings, based on the assignment given for BN fragments of varying lengths.[31]

Summary

■ INTRODUCTION

Vibrational spectroscopy [infrared absorption (IR) and Raman (RS)] is a widely used, reliable, and powerful method for studying conformational changes and molecular interactions and for unambiguously identifying and characterizing various molecules by their vibrational fingerprint. In the SERS spectrum of [Ala7]BN6−14 (Table 1) adsorbed on the AgNP surface (Figure 2B), the interactions between the indole ring of Trp[3] and the metal surfaces are indicated by bands at 1608 [W1, benzene + pyrrole and ν(N1−C8)], 1580 [W2], 1549 [W3, ν(C2 C3)], 1417 [W6, νs(N1C2C3) + δ(N1−H) and benzene δ(CH)], 1359/1341 [W7, indole ν(N1−C8); Fermi resonance], 1160 [δ(N1H)], 1011 [W16 (out-of-phase benzene ring breathing)], 876 [W17, δ(N1H) and Fermi resonance], and 758 cm−1 [W18, pyrrole ring breathing] These bands appear in the Raman and SERS spectra of all the peptides studied, except [Ala3]BN6−14, at similar wavenumbers but show different enhancement.

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES