عنوان

Studies on the binding of ovomucoid third domain to elastase:

TLpq304346050

انگلیسی

Studies on the binding of ovomucoid third domain to elastase:

[Thesis]

B. M. Baker

Towards the accurate prediction of binding energetics from structure

K. P. Murphy

The University of Iowa

1997

329

Ph.D.

The University of Iowa

1997

The binding of turkey ovomucoid third domain (OMTKY3) to porcine pancreatic elastase (PPE) has been studied using isothermal titration calorimetry (ITC) and empirical, structure-based thermodynamic calculations (structural energetics calculations). The aim of the studies is to provide new experimental data and the theoretical formalism necessary for the continued development of the structural energetics calculations. The calculated energetics are based upon a model of the PPE-OMTKY3 complex built using the crystallographic structures of both free components and the complex between human leukocyte elastase and OMTKY3. The calculations predict upon binding at 25C, a negligible usd\rm\Delta H\sp\circ,usd a large positive usd\rm\Delta S\sp\circ,usd and a large negative usd\rm\Delta C\sb{p},usd which yield a predicted usd\rm\Delta G\sp\circusd of 54 kT mol The ITC experiments indicate that binding of OMTKY3 to PPE is linked to a shift in the pKusd\rm\sb{a}usd of an ionizable group. Consequently, the observed binding energetics are strongly dependent upon solution conditions. Through a global analysis of experiments performed as a function of pH, buffer ionization enthalpy, and temperature, the intrinsic binding energetics have been determined, as have the energetics associated with the pKusd\rm\sb{a}usd shift. The experimental intrinsic binding energetics are in excellent agreement with those predicted from the calculations; the intrinsic usd\rm\Delta G\sp\circusd is usd\rm{-}60.6\pm0.5\ kJ\ mol\sp{-1}usd at 25C. Based on the composition of the protein-protein interface, published information, and the experimental measurements, we assign the pKusd\rm\sb{a}usd shift to the catalytic histidine (His57) in the PPE active site. The pKusd\rm\sb{a}usd shift is of particular interest. Its effects on the observed binding energetics highlight an important complication that can arise when comparing experimental and calculated energetics. The availability of the enthalpic and entropic components of the shift provides an excellent opportunity to study environmental effects upon ionization energetics. Finally, the pKusd\rm\sb{a}usd shift may be important for the enzymatic function of PPE. In summary, the PPE-OMTKY3 interaction has been studied in detail. In addition to providing new theoretical developments and experimental data, this work forms the foundation for future studies required for the further development of relationships between protein structure and energetics and the structural energetics calculations.

Biochemistry

Biochemistry

Biological sciences

Biophysics

pancreatic elastase

Pure sciences

B. M. Baker

K. P. Murphy

p

[Thesis]

276903

a

Y