o-Nitrotyrosine and p-iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes.
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 clear-cut 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.
De Filippis
Vincenzo
V
Department of Pharmaceutical Sciences, University of Padua, Italy. vincenzo.defilippis@unipd.it
Draghi
Annamaria
A
Frasson
Roberta
R
Grandi
Claudio
C
Musi
Valeria
V
Fontana
Angelo
A
Pastore
Annalisa
A
D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D010455:Peptides / Q000737:chemistry*; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D018909:src Homology Domains
D010455:Peptides; D011506:Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan