Papp, Robert (2002) Probing peptide binding to the second PDZ domain of hPTP1E using FT-IR and MS. Masters thesis, Concordia University.
Three peptides were evaluated for binding to the second PDZ domain of human protein tyrosine phosphatase 1E using FT-IR and mass spectrometry. This PDZ is known to bind the C-terminal motif X-Ser/Thr-Val. MS demonstrated the formation of a 1:1 complex between PDZ and ENEQVSAV but not with peptides ENEQVCAV and KDDEVYYV due to substitution of Ser in -2 position with Cys or Tyr. Furthermore, the H/D exchange rate of the PDZ-ENEQVSAV, monitored by MS, was reduced compared to free PDZ, suggesting a stable protein-ligand complex had formed. Thermal denaturation of free PDZ and in combinations with three peptides was investigated using FT-IR and CD spectroscopy. The T m of free PDZ in D 2 O was found to be 47 ± 1C̕ when monitoring the loss of Ý-sheets and the appearance of aggregation by FT-IR. Combinations of PDZ with KDDEVYYV, ENEQVCAV and ENEQVSAV revealed T m values of 48 ± 1, 49 ± 1 and 57 ± 1C̕, respectively. The increased T m observed with PDZ-ENEQVSAV was attributed to the formation of a stable complex. The thermal denaturation spectra were evaluated by 2D correlation analysis to determine the sequence of unfolding events. Ý-sheets and turns unfold prior to protein aggregation in free PDZ while in the PDZ-ENEQVSAV complex Ý-sheets unfold prior to aggregation but turns unfold last, indicating that residual turn-like structures persisted in the denatured state. Evidence from CD spectroscopy suggested that helical structures remained intact during heating to 75C̕ for PDZ and the PDZ-ENEQVSAV complex.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (Masters)|
|Pagination:||xii, 101 leaves : ill. ; 29 cm.|
|Degree Name:||Theses (M.Sc.)|
|Program:||Chemistry and Biochemistry|
|Thesis Supervisor(s):||English, Ann M.|
|Deposited By:||Concordia University Libraries|
|Deposited On:||27 Aug 2009 17:20|
|Last Modified:||08 Dec 2010 15:21|
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