Thermodynamic and kinetic analysis of the Escherichia coli thioredoxin-C' fragment complementation system.
Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a long alpha-helix. The two fragments, 1-37 and 38-108, were purified and characterized by using CD and fluorescence spectroscopy. Both fragments lack structure at neutral pH and room temperature. The secondary and tertiary structural contents of the non-covalent complex formed on the mixing of the two peptide fragments are 47% and 35% of the intact protein respectively. The thermodynamics and kinetics of fragment association were characterized by titration calorimetry and stopped-flow fluorescence spectroscopy. Single phases were observed for both association and dissociation, with rate constants at 298 K of kon=4971+/-160 M-1.s -1 and koff=0. 063+/-0.009 s-1 respectively. The ratio kon/koff was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process. The values for DeltaCp, DeltaH0 and DeltaG0 at 298 K for dissociation of the complex were 5.7 kJ. mol-1.K-1, 45.3 kJ.mol-1 and 29.8 kJ.mol-1 respectively. The value for DeltaH0 was linearly dependent on temperature from 8-40 degrees C, suggesting that DeltaCp is independent of temperature. The values for DeltaCp and DeltaG0 are very similar to the corresponding values for the unfolding of intact thioredoxin at 25 degrees C. However, both DeltaH0 and DeltaS are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.
Ghoshal
A K
AK
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Swaminathan
C P
CP
Thomas
C J
CJ
Surolia
A
A
Varadarajan
R
R
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*; D017186:Titrimetry; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D010446:Peptide Fragments; D014443:Tyrosine; D013879:Thioredoxins; D014364:Tryptophan; D003488:Cyanogen Bromide