Recent molecular dynamics simulations have suggested important roles for nanoscale dewetting in the stability, function, and folding dynamics of proteins. Using a synergistic simulation-experimental approach on the αTS TIM barrel protein, we validated this hypothesis by revealing the occurrence of drying inside hydrophobic amino acid clusters and its manifestation in experimental measures of protein stability and structure. Cavities created within three clusters of branched aliphatic amino acids [isoleucine, leucine, and valine (ILV) clusters] were found to experience strong water density fluctuations or intermittent dewetting transitions in simulations. Individually substituting 10 residues in the large ILV cluster at the N-terminus with less hydrophobic alanines showed a weakening or diminishing effect on dewetting that depended on the site of the mutation. Our simulations also demonstrated that replacement of buried leucines with isosteric, polar asparagines enhanced the wetting of the N- and C-terminal clusters. The experimental results on the stability, secondary structure, and compactness of the native and intermediate states for the asparagine variants are consistent with the preferential drying of the large N-terminal cluster in the intermediate. By contrast, the region encompassing the small C-terminal cluster experiences only partial drying in the intermediate, and its structure and stability are unaffected by the asparagine substitution. Surprisingly, the structural distortions required to accommodate the replacement of leucine by asparagine in the N-terminal cluster revealed the existence of alternative stable folds in the native basin. This combined simulation-experimental study demonstrates the critical role of drying within hydrophobic ILV clusters in the folding and stability of the αTS TIM barrel. © 2013 American Chemical Society.