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

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 (BN 6–14 ) 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.

Spectroscopic characterization and in vitro studies of biological activity of bradykinin derivatives

“…Intracellular inositol monophosphate (IP-one) assay. Receptor function was determined by measuring intracellular inositol monophosphate concentration using the IP-one kit from Cis-bio according to the procedure described previously (see Supporting Information for details) 21 . R 892 and WIN 64,338 hydrochloride (both from Tocris) were used as reference antagonists for B1R and B2R, respectively 51 .…”

“…These issues need to be addressed to better understand the interaction mechanism between BK and the receptors and are critical for the development of new BK antagonists with increased affinity for the receptors. Surface-enhanced Raman scattering (SERS) has shown promise in structural-activity studies to predict the biochemical activity of some neuropeptides [19][20][21] . For example, SERS has been used to determine the adsorbed molecular structures and the changes in these structures and adsorption resulting from substitution of natural amino acids by synthetic amino acids of bombesin (BN) and its analogs deposited on the Ag surface 19 .…”

Spectroscopic characterization and in vitro studies of biological activity of bradykinin derivatives

“…Intracellular inositol monophosphate (IP-one) assay. Receptor function was determined by measuring intracellular inositol monophosphate concentration using the IP-one kit from Cis-bio according to the procedure described previously (see Supporting Information for details) 17 . R 892 and WIN 64338 hydrochloride (both from Tocris) were used as reference antagonists for B1R and B2R, respectively.…”

“…These issues need to be addressed to better understand the interaction mechanism between BK and the receptors and are critical for the development of new BK antagonists with increased affinity for the receptors. Surface-enhanced Raman scattering (SERS) has shown promise in structure-activity studies to predict the biochemical activity of some neuropeptides 15,16,17 . For example, SERS has been used to determine the adsorbed molecular structures and the changes in these structures and adsorption resulting from the substitution of the natural amino acids with synthetic amino acids of bombesin (BN) and its analogs deposited on the Ag surface 15 .…”

‘Exploration and Expansion’ in Peptide Sequence: Discovery of Structurally-Optimized Polymyxin Derivatives Facilitated by Peptide Scanning and in situ Screening Chemistry

“…19 As for the determination of the modification site on peptides, a sophisticated methodology has been developed: peptide scanning. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] In this methodology, the systematic substitution of each amino acid in the peptide sequence for a certain scanning unit is used to investigate the importance of the corresponding amino acid residue.…”