| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
To evaluate contributions from Coulomb interactions between ionic charges on the surface of protein molecules to their structural stability, we have made both computational and experimental studies following the work of last year :
(1)Computational studies : For electrostatic mutants whose ionic charges on the molecular surface are partially neutralized, differential Poisson-Boltzmann equations were solved to estimate their Coulomb-interaction energies U_<c-> Applying this 'Partial Neutralization Analysis' method to cytochrome c and ubiquitin, we obtained following results :
(1)At neutral pH and low ionic strength, the average value U of U_c taken over the molecular species of partially neutralized tuna cytochrome c decreases initially, reaches a minimum at n=6 with+3 net charges, and then increases with increasing neutralization number n. This prediction agrees well with the observation that cytochrome c molecules change their conformation from the unfolded to the MG form with increasin
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g n. c At high ionic strength, the distribution of U_c has three distinct peaks, and U increases monotonously with n, This is caused by the fact that the contribution from interactions between nearby ionic residues, especially those of the two ion pairs, Lysl3-Glu9O and Lys53-Asp5O, dominate the Coulomb energy due to the shielding effect of salt ions. (3)Neutralizing acidic and basic residues of ubiquitin was found to have similar effects on the distribution of U_c.
(2)Experimental studies : First, we prepared partially neutralized samples of cytochrome c and purified them with respect to n. Using a high precision differential scanning calorimeter(DSC), we have measured heat capacity functions C_p(T)for the samples with n = 3-5. (1)The heat-absorption peak of C_p(T)due to thermal transition of modified proteins is broader than that of natural cytochrome c. This wil1 originate from the fact that, in the ensemble of neutralized molecules, there exist various molecular species that are neutralized at different sites and have different electrostatic energies. (2)The value of C_p(T)decreases above the temperature of thermal transition, which suggests that some intramolecular cohesive interactions are made as it is nearly independent of protein concentration. Less
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