Mutation of the hydrophobic residue on helix alpha5 of the Bacillus thuringiensis Cry4B affects structural stability.
Cry4B toxin is a mosquito-larvicidal protein from the Bacillus thuringiensis subsp. israelensis. We have investigated the role of two conserved hydrophobic residues of Cry4B in structural stabilization. Substitutions of the leucine-175 and isoleucine-189 on helix alpha5 with valine and leucine did not affect the expression level, solubility and proteolytic processing. Steady state analysis of an unfolding experiment as monitored by circular dichroism and fluorescence spectroscopy demonstrated a typical two-state transition. The determined unfolding free energy for the L175V mutant revealed a structural destabilization of 10.49 kcal/mol relative to the wild type. However unfolding kinetic analysis gave identical activation energy for wild type and both mutants. Our findings suggested that a perturbation on the close packing of the hydrophobic side chains in protein interior could lead to a significant destabilization of the native conformation.
Krittanai
Chartchai
C
Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phuttamonthon 4 Road, Nakhonpathom, 73170, Thailand. stckt@mahidol.ac.th
Bourchookarn
Apichai
A
Pathaichindachote
Wanwarang
W
Panyim
Sakol
S
D019943:Amino Acid Substitution; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001427:Bacterial Toxins; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics*; D006460:Hemolysin Proteins; D057927:Hydrophobic and Hydrophilic Interactions; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D007532:Isoleucine; D007930:Leucine