The energetics of protein folding and application to protein toxin mode of action

2.3.1 Folding of Soluble Proteins

The folding of soluble proteins has been studied by both chemical and temperature denaturation detected by numerous biophysical methods. For many soluble proteins, the folding is two-state, i.e., only native and denatured protein is observed. The folding reaction can be written as:

where N denotes the native protein conformation and D represents the denatured or "unfolded" form of the protein. The free energy associated with the unfolding, AG, can then be determined from the following relationship:

where [D] is the concentration of the denatured protein and [N] is the concentration of native protein. However, there are also many examples where intermediates in the folding are observed. Depending upon the specific protein, multiple intermediates may be observed and the folding reaction modified thusly:

where 11 represents the first observed intermediate, 12 the second observed intermediate, and so on through the nth intermediate. In the above representation, all intermediates are "on-pathway" toward denaturation, i.e., they are steps from folded to denatured protein states. However, this is not always the case as some intermediates may lead to off-pathway states, such as aggregates that are observed in the formation of protein fibrils. In the course of studying protein denaturation, another specific intermediate state was identified, the so-called molten globule (MG). The MG state is defined as a loss of tertiary structure without appreciable loss of secondary structure. This MG state has been identified in the denaturation of many proteins. The energetic stability of this state must be intermediate relative to the folded and unfolded states. Many studies have revealed that a state of the protein in which much tertiary structure is lost may play a critical role in the action of protein toxins. This point will be expanded below.

2.3.2 Folding of Membrane Proteins

The folding of membrane proteins can also be treated formally, like their water-soluble counterparts, where the unfolded state is replaced with the membrane-inserted state. Given the technical difficulties of studying the energetics of membrane insertion, the number of examples to date is limited. The pioneering studies of Popot and Engelman on membrane protein folding dealt with the folding and insertion of helical segments, where the process is essentially divided into helix folding and helix insertion.15 These types of studies have also been applied to the folding of integral membrane b-sheet proteins, particularly outer membrane proteins from E. coli.16 More recently, the White laboratory has developed methodologies and thermodynamic models for the folding and insertion of helical proteins into mem-branes.17 Those studies have initiated the dissection of the individual contributions of residues to the energetics of membrane helix insertion. One of the important conclusions of those studies is that the energetic barrier for the partitioning of unfolded or nonhydrogen-bonded peptide chains is very high and may be considered thermo-dynamically forbidden.

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