Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein.
The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.
Ganesh
C
C
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Shah
A N
AN
Swaminathan
C P
CP
Surolia
A
A
Varadarajan
R
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D006146:Guanidines; D058619:Maltose-Binding Proteins; D008320:Maltose; D019791:Guanidine