Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV.
Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) 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 2M GnHCl and 4M 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 (lambda(max)), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M 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.
Rasheedi
Sheeba
S
Laboratory of Molecular and Cellular Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Andhra Pradesh 500 076, India.
Suragani
Madhuri
M
Haq
Soghra K
SK
Ghosh
Sudip
S
Ehtesham
Nasreen Z
NZ
Hasnain
Seyed E
SE
D020106:Acrylamide / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D055592:Biophysical Phenomena / Q000187:drug effects; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification; D013696:Temperature; D014508:Urea / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; D020106:Acrylamide; D014508:Urea; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases; D019791:Guanidine