Comparative Study of Thermostability and Structure of Close Homologues - Barnase and Binase

Abstract

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

Thermodynamics of barnase unfolding

“…3). This value is somewhat larger than reported by Makarov et al (1993), 3.8 kJ K-I mol", but is in good correspondence with the denaturation heat capacity increment determined directly from the calorimetrically recorded heat capacity function at T, = 50 "C, the averaged value of which is 5.7 kJ K" mol" ( Table 2). The difference between the heat capacities of the native and dena- tured states depends on temperature (Fig.…”

“…In solutions close to neutral pH, reversible unfolding of barnase by denaturants and temperature appears to be a 2-state transition (Hartley, 1968(Hartley, , 1975Makarov et al, 1993). Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition.…”

“…Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition. However, later in 1993 the paper of Makarov et al (1993) ing subdomains. Analysis of the 3-dimensional structure of these proteins does indeed reveal 2 hydrophobic clusters (see Kinemage 1;Serrano et al, 1992).…”

“…Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition. However, later in 1993 the paper of Makarov et al (1993) appeared, with calorimetric observations of asymmetry in the melting profile of barnase at low pH values. The asymmetry in melting profile might indicate presence of a stable intermediate in unfolding, perhaps involving fold-ing subdomains.…”

“…Therefore, the mode of barnase denaturation required detailed investigation. Because there was also some deviation between our estimates of the enthalpy of denaturation and that of Makarov et al (1993), we felt it necessary to repeat calorimetric measurements on barnase to exclude any ambiguity in the thermodynamics of this protein. Figure 1 shows the temperature dependence of the partial molar heat capacity of barnase upon heating in acetate and glycine solutions of various pH.…”