Thermodynamics of barnase unfolding

Abstract

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 "C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features -the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.Keywords: barnase; hydrogen bonding; hydrophobic interactions; scanning microcalorimetry Barnase attracts the special interest of researchers engaged in studying protein folding because it is one of the smallest globular proteins (1 10 amino acid residues; see Kinemage 1) that does not have disulfide crosslinks but is quite stable and unfolds reversibly. The latter circumstance is very important for quantitative analysis of this process by equilibrium thermodynamics, specifying the stability of the native state in energetic terms.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 Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is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. 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). 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 ...

Thermodynamics of denaturation of mutants of barnase with disulfide crosslinks

“…This pro®le and the magnitude of the protonation changes are in excellent agreement with earlier work (Oliveberg et al, 1994). Similarly, the value of ÁG D-N of 10.2 kcal mol À1 at 298 K for wild-type barnase at near neutral pH is close to estimates obtained in other studies (Griko et al, 1994;Martinez et al, 1994;Oliveberg et al, 1994;Johnson & Fersht, 1995). The value obtained from urea equilibrium unfolding of barnase at this temperature is somewhat lower and the reasons for this discrepancy have been examined elsewhere (Johnson & Fersht, 1995).…”

“…Thermal unfolding of barnase DSC and circular dichroism (CD) data for the thermal denaturation of barnase are of a similar or higher quality to that published from our group earlier (Matouschek et al, 1994;Oliveberg et al, 1994;Johnson & Fersht, 1995 (Griko et al, 1994;Martinez et al, 1994;Matouschek et al, 1994;Johnson & Fersht, 1995).…”

“…Barnase is also suited to such studies since its thermal unfolding is fully reversible and characterised by a two-state transition with a large enthalpy change. Several studies have used differential scanning calorimetry (DSC) to examine the thermodynamics of the wild-type protein and a limited number of mutants (Griko et al, 1994;Martinez et al, 1994;Matouschek et al, 1994;Johnson & Fersht, 1995). Here, we report the thermodynamic characterisation of mutants of barnase with disul®de crosslinks as well as the corresponding single and double cysteine mutants obtained under reducing conditions.…”

Molecular dynamics study of the interaction of Aβ(13-23) with β-sheet inhibitors

“…41 Calorimetric measurements on barnase yielded an estimate of 12 kJ/mol. 42 In globular proteins, an estimate of 9.2 kJ/mol has been determined, 43 similar to the value in human lysozyme, 8.9 ± 2.6 kJ/mol. 44 However, a survey of all classes of proteins yielded a much lower value, 0.95 kJ/mol.…”

Hydrophobic folding units derived from dissimilar monomer structures and their interactions

“…Fourth, for the case of barnase, the three clearly discerned hydrophobic clusters correspond to a single hydrophobic folding unit. This is.consistent with the experimental evidence showing barnase to follow a two-state folding model (Griko et al, 1994). Incorrectly parsing barnase into three independent units would have resulted in too much exposed non-polar surface area at their interface, rendering these subdomain-nucleation centers unstable entities.…”