Protein thermal denaturation, side-chain models, and evolution: amino acid substitutions at a conserved helix-helix interface

Abstract

Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries. Mutants with these combinations are biologically functional. Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants. Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically. Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions. First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently. Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

“…The value of ⌬C p has been reported to change for mutated proteins (28,31). Due to the uncertainty associated with determining the value of ⌬C p for each of the mutant proteins, the change in free energy of denaturation was calculated assuming that ⌬H and ⌬S are independent of temperature which requires only the apparent enthalpy of the wild type protein and the T m of the wild type and mutant proteins.…”

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

“…A major structural determinant of apocytochrome c 2 for binding CcmI is its most C-terminal helix, the equivalent of which is conserved in most class I c-type cytochromes (55). This helix packs orthogonally against the heme-binding site containing N-terminal helix in mature c-type cytochromes.…”

A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

“…Also in support of the model, the structures of a-helical coiled coils appear to be determined by the shapes of the buried side chains (6). In contrast with this view, it has been shown that alternative core sequences that lead to viable proteins could be selected by random mutagenesis for both A-repressor (11) and T4 lysozyme (12), among others (13,14). It is possible, however, that a limited number of combinations of amino acids are viable and that they are the ones identified by the mutagenic selection.…”