The energetic effects of \core mutations in T4 lysozyme (blue) and bacteriorhodopsin (red) as a function of cavity size increases. Cavities were measured in terms of volume (left panel) and surface area (right panel). The data for T4 lyso-zyme were taken from Eriksson et al.12 For bacteriorhodopsin, multiple conformations were observed for side chains in the newly created cavity in some cases. Filled symbols represent the cavity size using only the wild-type conformation and the open symbols are from the weighted average using all alternate conformations. Circles indicate results for the L-to-A or I-to-A mutants that delete three carbons. Triangles indicate results for the V49A and I148V that delete different numbers of carbons. The line was fit to only the four L-to-A or Ito- A substitutions, using the wild-type rotamers only (filled circles). Horizontal error bars indicate the standard deviation of the cavity size changes using three independent bacteriorhodopsin structures: 1C3W 13, 1PY6 6 and 1XJI 14. Vertical error bars indicate the standard deviation of triplicate stability measurements. The data are provided in Tables S2–S4 in the Supporting Information.

Comparison of the wild type and mutant bacteriorhodopsin structures. Residues within 4 Å of altered side chains are shown in stick representation and the mutant side chain, labeled in red, in ball and stick representation. The wild-type protein structure (PDB ID: 1PY6, Chain B) is shown in CPK colors and the mutant structure is shown in red. Omit electron density is shown contoured at 1.0 σ revealing no density for the deleted atoms. The residues that adopt altered conformations in the L111A, I148A and I148V proteins are labeled in black.