High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clearcut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner.Keywords: SH3 domains; proline-rich peptides; o-nitrotyrosine; p-iodophenylalanine; protein recognition; noncoded amino acids; fluorescence energy transfer; quenching; circular dichroism Supplemental material: see www.proteinscience.orgThe central role played by molecular interactions in the functioning of all biological systems requires a detailed structural understanding of how recognition takes place (Jones and Thornton 1996;Pawson and Nash 2003). Complete structural determination of protein complexes can, however, be time-demanding, since with the current technical tools it is often necessary to undertake de novo structure determinations even when the structures of all the interacting partners are known individually. It is therefore increasingly important to develop new high-throughput approaches that may allow us to grasp quickly at least the basic features of a complex without its detailed and lengthy structural description. Abbreviations: Standard single-or three-letter abbreviations were used for natural amino acids; CD, circular dichroism; DIEA, diisopropylethylamine; EDT, ethanedithiol; ESI, electrospray ionization; Fmoc, 9-fluorenyl-methyloxycarbonyl; FRET, fluorescence resonance energy transfer; IF, p-iodophenylalanine; IFE, inner filter effect; HATU, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; Myo3, isoform 3 of myosin from the yeast Saccharomyces cerevisiae; Myo3-SH3, SH3 domain of Myo3;