Myristoylation, the covalent linkage of a saturated, C 14 fatty acyl chain to the N-terminal glycine in a protein, plays a vital role in reversible membrane binding and signaling by the modified proteins. Currently, little is known about the effects of myristoylation on protein folding and stability, or about the energetics and molecular mechanisms of switching involving states with sequestered versus accessible myristoyl group. Our analysis of these effects in hisactophilin, a histidine-rich protein that binds cell membranes and actin in a pH-dependent manner, shows that myristoylation significantly increases hisactophilin stability, while also markedly increasing global protein folding and unfolding rates. The switching between sequestered and accessible states is pH dependent, with an apparent pK switch of 6.95, and an apparent free energy change of 2.0 kcal·mol −1 . The myristoyl switch is linked to the reversible uptake of ∼1.5 protons, likely by histidine residues. This pH dependence of switching appears to be the physical basis of the sensitive, pH-dependent regulation of membrane binding observed in vivo. We conclude that an increase in protein stability upon modification and burial of the attached group is likely to occur in numerous proteins modified with fatty acyl or other hydrophobic groups, and that the biophysical effects of such modification are likely to play an important role in their functional switches. In addition, the increased global dynamics caused by myristoylation of hisactophilin reveals a general mechanism whereby hydrophobic moieties can make nonnative interactions or relieve strain in transition states, thereby increasing the rates of interconversion between different states. thermodynamic cycle | switch dynamics | switch energetics M yristoylation is a common cotranslational modification found in ∼0.5-0.8% of eukaryotic proteins (1). This modification involves the covalent linkage of a saturated C 14 fatty acyl chain to the N-terminal glycine residue in a protein (1). Myristoylated proteins play vital roles in many biological processes and commonly undergo reversible switches. The "flipping" of myristoyl switches typically involves interconversion between a myristoyl-sequestered state, myr seq , where the myristoyl group is located in a hydrophobic binding pocket within the protein, and a myristoyl-accessible state, myr acc , where the myristoyl group is available for binding to membranes or other proteins. Switching may be associated with relatively large or subtle structural and/or dynamic changes in the myristoylated protein (2, 3). It can also be regulated by binding of various ligands (e.g., H þ , Ca 2þ , GTP, or regulatory protein) (3-5). Some examples of proteins that undergo myristoyl switching include: Ca 2þ -dependent recoverin, which mediates photoresponses in the retina (3); Ca 2þ -dependent guanylate cyclase activating protein (GCAP), which regulates the function of guanylate cyclase (2, 6); oligomerization-dependent HIV-1 Gag, which orchestrates HIV-1 viral proliferat...