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Probing peptide binding to the second PDZ domain of hPTP1E using FT-IR and MS

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Probing peptide binding to the second PDZ domain of hPTP1E using FT-IR and MS

Papp, Robert (2002) Probing peptide binding to the second PDZ domain of hPTP1E using FT-IR and MS. Masters thesis, Concordia University.

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Abstract

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)
Authors:Papp, Robert
Pagination:xii, 101 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (M.Sc.)
Program:Chemistry and Biochemistry
Date:2002
Thesis Supervisor(s):English, Ann M.
ID Code:1553
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:20
Last Modified:08 Dec 2010 10:21
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