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...

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin-and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.coarse-grained simulation | funnel landscape | β-trefoil P rotein folding is governed by various physicochemical forces that bias the native state, which for many proteins is also the functional state, over the many alternative nonnative states. The network of native interactions has been found in many cases to be sufficient to capture the folding mechanism and kinetics of proteins (1, 2). The discrimination between native and nonnative interactions is the foundation of the principle of minimal frustration (3) and explains the power of native topology-based models in studying folding biophysics (4). The dominant role of native interactions is manifested by the funnel-shaped energy landscape for folding that suggests folding is robust and an efficient process. The information stored in the native topology may, however, be tuned by various factors such as confining the protein in a small space, crowding agents, or conjugating the protein to other biomolecules [e.g., oligosaccharides (5) or fatty acyl chains such as myristoyl (6)]. In addition to manipulating folding characteristics by modifications or environmental conditions, nonnative interactions, which are by definition in conflict with the native state, may decorate the folding funnel (7, 8) by increasing energetic frustration (9) between interactions and therefore landscape roughness. The degree of roughness, which affects the trapping of the protein in nonnative states, depends on the particular sequence of the pr...

Advanced cryo–electron microscopy yields high-resolution structures of proteins

Although the majority of natural proteins exist as protein-protein complexes, the molecular basis for the formation and regulation of such interactions and the evolution of protein interfaces remain poorly understood. We have investigated these phenomena by characterizing the thermal and chemical denaturation of thermophilic DsrEFH proteins that have a common subunit fold but distinct quaternary structures: homodimeric Tm0979 and homotrimeric Mth1491. Tm0979 forms a moderate affinity dimer, and a monomeric intermediate is readily populated at equilibrium and during folding kinetics. In contrast, the Mth1491 trimer has extremely high stability, so that a monomeric form is not measurably populated at equilibrium, although it may be during folding kinetics. A common mechanism for evolution of quaternary structures may be facile formation of a relatively stable monomeric species, with stabilizing intermolecular interactions centering on alternative environments for a beta-strand at the edge of the monomer, augmented by malleable hydrophobic interactions. The exceptional trimer stability arises from a remarkably slow unfolding rate constant, 6.5 x 10(-13) s(-1), which is a common characteristic of highly stable thermophilic and/or oligomeric proteins. The folding characteristics of Tm0979 and Mth1491 have interesting implications for assembly and regulation of homo- and heterooligomeric proteins in vivo.

The energetic networks that govern regulated switching processes in macromolecules are poorly understood at a molecular level. We illustrate a general methodology that uses thermodynamic cycles to measure the coupling energetics between specific groups in a macromolecule and ligand-binding-induced macromolecular switching. The approach is applied to new and published thermodynamic stability and/or binding data not previously analyzed in this way, for a wide range of switching systems, including H+ or Ca²+-binding-induced myristoyl switching, ion or peptide-binding-induced conformational switching in various proteins and small molecule binding to a ribo-switch. The results show how this powerful approach can be used to identify and dissect the molecular determinants of switching in macromolecules.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.