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 ⌽-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 ⌽ values >0.8. However, residues Y43 and T46 give lower ⌽ 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.phi value ͉ kinetics ͉ thermodynamics ͉ protein engineering P rotein folding plays a central role in biology. Folding investigations provide key information on protein structure and dynamics, while protein misfolding can have serious disease implications (1, 2). To fold correctly, proteins must overcome an activation barrier to pass through a high-energy transition state. Understanding the nature of this folding transition state is important in resolving how a protein folds to a stable and functional native structure (3, 4).The most powerful method available to probe the structure and energetics of the folding transition state combines sitedirected mutagenesis, equilibrium thermodynamics, and kinetic data in a ⌽-value analysis. This approach has revolutionized protein folding studies by providing a quantitative description of the environment experienced by individual side chains in the folding transition state and has been applied to the folding of many small, water-soluble proteins (3,(5)(6)(7)(8). However, the ⌽-value method has yet to be applied to larger integral membrane proteins.Integral membrane proteins are a special case in protein folding because they are adapted to the lipid bilayer rather than to the cytoplasmic milieu (9). The sequences of transmembrane regions are biased in favor of hydrophobic amino acids to match the low dielectric of the membrane interior. This...