title: ESI-MS and FTIR studies of the interaction between the second PDZ domain of hPTP1E and target peptides.
abstract: The specificity of interaction between the second PDZ domain of human protein tyrosine phosphatase1E (PDZ2) and a C-terminal peptide, ENEQVSAV, from the guanine nucleotide exchange factor RA-GEF-2 was investigated using Fourier transform infrared (FTIR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Specificity of the binding interaction and the importance of Ser in the -2 position of the target peptide were demonstrated using alternate peptides ENEQVCAV and KDDEVYYV. FTIR-monitored thermal denaturation in the amide I region showed a 10 degrees C increase in melting temperature (Tm) for the PDZ2-ENEQVSAV complex compared with that of free PDZ2, and the spectra revealed increased absorption in the beta-sheet region (1628 cm(-1)) of PDZ2 on peptide binding. Neither of these results were observed with peptides containing either Cys or Tyr in the -2 position. Complex formation with the Ser-containing peptide was further demonstrated by direct measurement of a 1:1 PDZ-peptide complex by ESI-MS in 100% aqueous solutions without the need for organic co-solvents. Our results demonstrate that even a single atom (O --> S) substitution from Ser to Cys in the -2 position disrupts C-terminal peptide binding to PDZ2.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
authors.forename: Robert
authors.initials: R
authors.lastname: Papp
authors.forename: Irena
authors.initials: I
authors.lastname: Ekiel
authors.forename: Ann M
authors.initials: AM
authors.lastname: English
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; C485746:RAPGEF6 protein, human; D011994:Recombinant Proteins; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases