|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1990 : ¥900,000 (Direct Cost : ¥900,000)
To study the role of Pro residues in the conformation, stability, and function of a protein, nine mutant alpha-subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six conserved Pro residues(positions 28, 57, 62, 96, 132 and 207)in 10 microorganisms, were constructed.
1) The far-UV CD spectra of five mutant alpha-subunits with Ala in place of Pro, the exception being the mutant at position 207(P207A), were identical to the spectrum of the wild-type protein. CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha-subunit.
2)Scanning calorimetric measurements(DASM4)showed that the stability of each mutant protein relative to that of the wild-type was about the-same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A ; which are substituted at less mobile positions.
3)To understand how the alpha and beta_2 subunits of tryptophan synthase interact to form an alpha_2beta_2 complex and undergo mutual activation, we have investigated isothermal calorimetric titrations (Omega) of wild type beta_2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of Gly for Pro at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. We conclude that Pro 132 plays a critical role in subunit interaction and in mutual subunit activation.