Mapping the tropomyosin isoform 5 binding site on human erythrocyte tropomodulin: Further insights into E-Tmod/TM5 interaction
Section snippets
Generation of human E-Tmod deletion clones
Plasmid pMALc2-MBP/Tmod [24], which encodes a maltose-binding protein (MBP) and a full-length human E-Tmod cDNA was digested with EcoRI followed by nuclease S1 to create blunt ends. The bidirectional exonuclease III digestion was stopped by adding 5 mM EDTA at different time points (10 s to 3 min). The cDNAs were filled in with Klenow fragment and religated to generate a nested set of deletion clones. Two additional clones were obtained by BamHI and PstI digestion, respectively, followed by
hTM5 binding to truncated E-Tmod
To map the hTM5 binding site, 35 human E-Tmod deletion clones truncated from the C-terminus were generated. They contain E-Tmod residues 1–334, -329, -325, -319, -318, -311, -306, -300, -299, -297, -294, -288, -279, -277, -276, -274, -271, -266, -263, -257, -249, -232, -221, -212, -197, -193, -179, -176, -170, -161, -127, -104, -70, -46, and -0, respectively. Recombinant proteins in E. coli were induced and affinity purified, and their ability to bind to hTM5 was determined by
Discussion
In this study, we used recombinant E-Tmod clones truncated from the C-terminus to identify a small region (23 residues from 105 and 127 or 6% of 359 residues) that is essential to interact with the recombinant hTM5 as detected by immunoprecipitation. This region is narrower than the region (100 residues from 39 to 138, or 28%) previously mapped using E-Tmod clones truncated from the N-terminus that interacted with TMs purified from human erythrocyte membranes as detected by solid phase binding
Acknowledgments
This work was supported by NIH Research Grant PO1HL-43026-6 from the National Heart, Lung, and Blood Institute (Project 2, LAS). Carlos Vera was supported by a pre-doctoral fellowship from UCMEXUS-CONACYT and a postdoctoral fellowship from Sung’s Molecular Bioengineering Laboratory. We used the Biotech Core in the Department of Bioengineering established with the support by the Whitaker Foundation. We would like to express our appreciation for Dr. Russell Doolittle’s advice on hTM5 and E-Tmod
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