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Biophys. J. BioFAST: First Published March 30, 2007. doi:10.1529/biophysj.106.094961
© 2007 by the Biophysical Society.

A more recent version of this article appeared on June 15, 2007.
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biophysj.106.094961v1
92/12/4424    most recent
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PROTEINS

LOW-RESOLUTION STRUCTURE AND FLUORESCENCE ANISOTROPY ANALYSIS OF PROTEIN TYROSINE PHOSPHATASE {eta} CATALYTIC DOMAIN

Huita C. Matozo 1, Maria A. M. Santos 1, Mario de Oliveira Neto 1, Lucas Bleicher 1, Luís M.T.R. Lima 2, Rodolfo Iuliano 3, Alfredo Fusco 4 and Igor Polikarpov 5*

1 Instituto de Física de São Carlos, Departamento de Física e Informática
2 Faculdade de Farmácia, Universidade Federal do Rio de Janeiro
3 Facoltà di Medicina e Chirurgia, Università di Catanzaro
4 Facolta` di Medicina e Chirurgia, Universita` di Napoli
5 Instituto de Fisica de Sao Carlos, USP

* To whom correspondence should be addressed. E-mail: ipolikarpov{at}if.sc.usp.br.

Submitted on August 12, 2006
Revised on September 21, 2006
Accepted on 22 February 2007


  Abstract
The rat protein tyrosine phosphatase {eta}, rPTP{eta}, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTP{eta} and the human homolog DEP-1 are down-regulated in rat and human neoplastic cells, respectively. However, the malignant phenotype is reverted after exogenous reconstitution of rPTP{eta}, suggesting that its function restoration could be an important tool for gene therapy of human cancers. Using small-angle X-ray scattering (SAXS) and biophysical techniques, we characterized the intracellular catalytic domain of rat protein tyrosine phosphatase {eta} (rPTP{eta}CD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTP{eta}CD dimers are elongated and have an average radius of gyration of 2.65 nm and a Dmax of 8.5 nm. To further study the rPTP{eta}CD conformation in solution, we built rPTP{eta}CD homology models using as scaffolds the crystallographic structures of RPTP{alpha}-D1 and RPTPµ-D1 dimers. These models were, then, superimposed onto ab initio low-resolution SAXS structures. The structural comparisons and sequence alignment analysis of the putative dimerization interfaces provide support to the notion that the rPTP{eta}CD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTP{alpha}-D1 dimer than to the dimeric arrangement exemplified by RPTPµ-D1. Finally, the characterization of rPTP{eta}CD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 ± 2.0 µM

Key Words: SAXS, fluorescence anisotropy, homology model, low-resolution shape, protein tyrosine phosphatases, solution structure






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Copyright © 2007 by the Biophysical Society.