Biology relies on the precise self-assembly of its molecular components. Generic principles of protein folding have emerged from extensive studies on small, water-soluble proteins, but it is unclear how these ideas are translated into more complex situations. In particular, the one-third of cellular proteins that reside in biological membranes will not fold like water-soluble proteins because membrane proteins need to expose, not hide, their hydrophobic surfaces. Here, we apply the powerful protein engineering method of Phi-value analysis to investigate the folding transition state of the alpha-helical membrane protein, bacteriorhodopsin, from a partially unfolded state. Our results imply that much of helix B of the seven-transmembrane helical protein is structured in the transition state with single-point alanine mutations in helix B giving Phi values >0.8. However, residues Y43 and T46 give lower Phi values of 0.3 and 0.5, respectively, suggesting a possible reduction in native structure in this region of the helix. Destabilizing mutations also increase the activation energy of folding, which is accompanied by an apparent movement of the transition state toward the partially unfolded state. This apparent transition state movement is most likely due to destabilization of the structured, unfolded state. These results contrast with the Hammond effect seen for several water-soluble proteins in which destabilizing mutations cause the transition state to move toward, and become closer in energy to, the folded state. We thus introduce a classic folding analysis method to membrane proteins, providing critical insight into the folding transition state.
|Translated title of the contribution||The transition state for integral membrane protein folding|
|Pages (from-to)||773 - 778|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - Jan 2009|