Cartoon diagram depicting the states used to calculate amino acid insertion energies. (A) The total energy of a reference peptide harboring between zero and seven TM leucine residues in a background of TM alanine residues is calculated in solution (left) and then in the presence of the membrane (right). In both states, the three terms in Eq. 1 or four terms in Eq. 3 are calculated. Helices were constructed using MOLDA (Yoshida and Matsuura, 1997). (B) The central residue (green) was systematically replaced by all other residues, except proline, and the energy calculations between solution and membrane were repeated. Carefully subtracting energy values computed from B with those from the reference peptide in A removes contributions to the insertion energy from the background residues as discussed in the online supplemental material.