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Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from Ac NPV
Author(s) -
Rasheedi Sheeba,
Suragani Madhuri,
Haq Soghra K.,
Ghosh Sudip,
Ehtesham Nasreen Z.,
Hasnain Seyed E.
Publication year - 2009
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21180
Subject(s) - chemistry , circular dichroism , rna polymerase , folding (dsp implementation) , protein secondary structure , biophysics , fluorescence , quenching (fluorescence) , denaturation (fissile materials) , protein folding , rna , biochemistry , crystallography , gene , biology , nuclear chemistry , physics , quantum mechanics , electrical engineering , engineering
Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus ( Ac NPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non‐two state, biphasic transition. Intermediate states of LEF4 at 2 M GnHCl and 4 M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady‐state fluorescence and far‐UV CD showed that while there was considerable shift in the wavelength of emission maximum (λ max ), the secondary structure of LEF4 intermediates at 2 M GnHCl and 4 M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far‐UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 574–582, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com