Translational initiation factor IF2 from Bacillus stearothermophilus: a spectroscopic and microcalorimetric study of the C-domain.
Conformation and stability of the C-terminal domain of initiation factor IF2 from Bacillus stearothermophilus were analyzed by circular dichroism, fluorescence and Raman spectroscopy, and microcalorimetry under different solvent conditions. From circular dichroism and Raman measurements, IF2C at neutral pH can be classified as an alpha + beta protein. Solvent perturbation and Raman spectroscopy indicate a high accessibility of the tyrosine residues in the native protein. The Gdn/HCl-induced unfolding of IF2C was monitored by circular dichroism. IF2C unfolding at neutral pH proceeds in two discrete steps. The midpoints (c(m)) and the free energy of unfolding (deltaG(u)H2O) of the first and second transition are 2.05 M and 6.2 kcal x mol(-1) and 4.1 M and 12.9 kcal x mol(-1), respectively. ANS does not bind to the stable intermediate formed at 3 M Gdn/HCl. It seems likely that IF2C is composed of two subdomains which unfold in a stepwise process. Melting experiments at pH 7.0 are impaired by irreversible aggregation at higher temperatures. However, in Gdn/HCl containing buffer at denaturant concentrations up to 1.5 M the melting becomes a reversible process and can be analyzed by differential scanning calorimetry. At Gdn/HCl concentrations between 1.0 and 1.5 M, IF2C seems to be composed of two folding units with Tm values of about 60 and 78 degrees C and folding enthalpy values (deltaHm) of about 37 and 58 kcal x mol(-1). At pH values below pH 3.0, IF2C can adopt a new acid-induced conformation, which is characterized by a high secondary structure content and a strong ANS binding. The Gdn/HCl-induced unfolding of IF2C at pH 2.6 takes place only in one discrete step with a midpoint c(m) of 3.3 M and a deltaG(AUa)H2O of 11.9 kcal x mol(-1).
Misselwitz
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R
Institute of Biochemistry, Medical Faculty (Charite), Humboldt University, Berlin, Germany.
Welfe
K
K
Krafft
C
C
Gualerzi
C O
CO
Welfle
H
H
D002151:Calorimetry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D039881:Prokaryotic Initiation Factor-2; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D014443:Tyrosine
D006146:Guanidines; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D011994:Recombinant Proteins; D012997:Solvents; D014443:Tyrosine; D019791:Guanidine