The thermodynamics of protein-protein recognition as characterized by a combination of volumetric and calorimetric techniques: the binding of turkey ovomucoid third domain to alpha-chymotrypsin.
We have used ultrasonic velocimetry, high-precision densimetry, and fluorescence spectroscopy, in conjunction with isothermal titration and differential scanning calorimetry, to characterize the binding of turkey ovomucoid third domain (OMTKY3) to alpha-chymotrypsin. We report the changes in volume and adiabatic compressibility that accompany the association of these proteins at 25 degrees C and pH 4.5. In addition, we report the changes in free energy, enthalpy, entropy, and heat capacity upon the binding of OMTKY3 to alpha-chymotrypsin over a temperature range of 20-40 degrees C. Our volume and compressibility data, in conjunction with X-ray crytsallographic data on the OMTKY3-alpha-chymotrypsin complex, suggest that 454(+/-22) water molecules are released to the bulk state upon the binding of OMTKY3 to alpha-chymotrypsin. Furthermore, these volumetric data suggest that the intrinsic compressibility of the two proteins decreases by 7%. At each temperature studied, OMTKY3 association with alpha-chymotrypsin is entropy driven with a large, unfavorable enthalpy contribution. The observed entropy of the binding reflects interplay between two very large favorable and unfavorable terms. The favorable term reflects an increase in the hydrational entropy resulting from release to the bulk of 454 water molecules. The unfavorable term is related to a decrease in the configurational entropy and, consequently, a decrease in the conformational dynamics of the two proteins. In general, we discuss the relationship between macroscopic and microscopic properties, in particular, identifying and quantifying the role of hydration in determining the thermodynamics of protein recognition as reflected in volumetric and calorimetric parameters.
Filfil
Rana
R
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ont., Canada M5S 2S2.
Chalikian
Tigran V
TV
D000818:Animals; D002151:Calorimetry; D002918:Chymotrypsin / Q000378:metabolism*; D019281:Dimerization; D005454:Fluorescence Polarization; D008433:Mathematics; D010059:Ovomucin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014422:Turkeys; D014867:Water / Q000737:chemistry
D014867:Water; D010059:Ovomucin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin