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Title: Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

Abstract

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous {alpha} and {beta} subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length {lambda}CI of the pBT bait vector and also NifD-K (fusion) with the N-terminal {alpha}-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the {beta}-{beta} interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the {alpha}-RNAP of the pTRG vector and {lambda}CI protein of the pBTmore » vector. The strength of the interaction between the proteins in study was determined by measuring the {beta}-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the {beta}-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the {beta}-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.« less

Authors:
 [1];  [1];  [2]
  1. Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762 (United States)
  2. Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762 (United States). E-mail: gavini@biology.msstate.edu
Publication Date:
OSTI Identifier:
20713447
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 337; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2005.09.105; PII: S0006-291X(05)02040-1; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AZOTOBACTER; GALACTOSIDASE; GENES; HYBRIDIZATION; NITROGENASE

Citation Formats

Lahiri, Surobhi, Pulakat, Lakshmi, and Gavini, Nara. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.09.105.
Lahiri, Surobhi, Pulakat, Lakshmi, & Gavini, Nara. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein. United States. doi:10.1016/j.bbrc.2005.09.105.
Lahiri, Surobhi, Pulakat, Lakshmi, and Gavini, Nara. Fri . "Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein". United States. doi:10.1016/j.bbrc.2005.09.105.
@article{osti_20713447,
title = {Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein},
author = {Lahiri, Surobhi and Pulakat, Lakshmi and Gavini, Nara},
abstractNote = {The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous {alpha} and {beta} subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length {lambda}CI of the pBT bait vector and also NifD-K (fusion) with the N-terminal {alpha}-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the {beta}-{beta} interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the {alpha}-RNAP of the pTRG vector and {lambda}CI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the {beta}-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the {beta}-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the {beta}-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.},
doi = {10.1016/j.bbrc.2005.09.105},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 337,
place = {United States},
year = {Fri Nov 18 00:00:00 EST 2005},
month = {Fri Nov 18 00:00:00 EST 2005}
}