ProteinLogic (United Kingdom)

Potassium channels govern the permeability of cells to potassium ions, thereby controlling the membrane potential. In metazoa, potassium channels are encoded by a large, diverse gene family. Previous analyses of this gene family have focused on its diversity in mammals. Here we have pursued a more comprehensive study in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes. The investigation revealed 164 potassium channel encoding genes in C. elegans, D. melanogaster, and mammals, classified into seven conserved families, which we applied to phylogenetic analysis. The trees are discussed in relation to the assignment of orthologous relationships between genes and vertebrate genome duplication.

Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.

The conventional procedure for analyzing urea denaturation curves assumes that the free energy of unfolding (delta GU-F) is linearly related to [urea] that is, delta GU-F = delta GH2O(U-F)--m[urea], where m is a constant, specific for each protein, and delta GH2O(U-F) is the free energy of unfolding in water. This relationship can be measured directly, however, over only a small concentration range of approximately +/- 0.8 M urea around the midpoint of the unfolding transition. A nagging discrepancy (1.6 kcal mol-1) between delta GH2O(U-F) at 298 K of barnase extrapolated from such an equation and the equivalent value obtained from thermal unfolding measurements has stimulated a re-evaluation of the equation. Differential scanning calorimetric measurements have been made of the thermal unfolding of barnase in the presence of concentrations of urea between 0 and 4.5 M, the midpoint of the unfolding transition at 298 K, to test the denaturation equation over a wide range of [urea]. Values for delta GU-F at 298 K (delta G298U-F) for each concentration of urea were extrapolated from the calorimetrically measured enthalpies and the denaturational heat capacity change (delta Cdp) measured for that concentration of urea. A plot of delta G298U-F against [urea] deviates systematically from linearity and fits better the equation: delta G298U-F = 10.5 +/- 0.08 - ((2.65 +/- 0.05) x [urea]) + ((0.08 +/- 0.01) x [urea]2) kcal mol-1. The curvature in the plot leads to apparent values of m that increase when measurements are made at lower concentrations of urea. This could account for increases in m at low values of pH or in destabilized mutants since the protein denatures at lower concentrations of urea. It has been shown previously that small curvature in the free energy of unfolding versus [urea] leads to negligible errors in measurements of delta delta GU-F, the change in free energy of unfolding on mutation, providing that the curvature is similar for all mutants. The calorimetrically measured enthalpies of unfolding are decreased in the presence of urea while delta Cdp is increased. Both of these observations are consistent with an overall exothermic interaction between urea and protein with a net increase on unfolding.

Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of association of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies of the binding of fragments and from thermal denaturation of the complexes. In general, there is a good correlation between the changes on mutation of the free energy of association of fragments and the changes in free energy of folding of the uncleaved parent protein. The notable exceptions are for residues in regions of the fragments that form nonnative hydrophobic clusters in the isolated fragments; mutation of the hydrophobic residues involved in these clusters decreases the equilibrium constant for formation of the noncovalent complex less than it does the equilibrium constant for folding of intact protein. The dissociated fragments must be destabilized by mutation of those hydrophobic residues, but to a lesser extent than is the complex itself. These clusters are thus less important energetically in the denatured state of the intact protein. The second-order rate constants for the major phase of association change with mutation, similar results being obtained from fluorescence measurements of the regain of tertiary structure and from circular dichroism measurements of the regain of secondary structure. The rate constants for association correlate well, in general, with the rate constants of refolding of the respective uncleaved proteins.(ABSTRACT TRUNCATED AT 250 WORDS)