Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.protein folding | loop closure entropy | molecular dynamics R educing the difference in entropy between the unfolded and folded states can increase the thermodynamic stability of a protein. This is commonly accomplished by strategies that act to restrict the conformational space available for the unfolded state (e.g., decreasing loop length, macromolecular crowding, and backbone cyclization) (1). In principle, changes that increase the entropy of the folded state can also lead to its stabilization provided that they exceed the loss of enthalpic contributions, if stabilizing interactions are perturbed by the modifications.The current work originally aimed at studying the effects exerted by changes in the length of a loop region on protein stability and folding. As a model, we chose a loop in human muscle acylphosphatase (hmAcP), a small (∼100 aa) enzyme that catalyzes the hydrolysis of the carboxyl-phosphate bond in various acylphosphate compounds and presents an open α/β-sandwich structure (ref. 2, and see, e.g., refs. 3-5). The folding stability and dynamics of hmAcP have been studied extensively and are well characterized (ref. 6-10 and references therein). Excluding a minor cis-trans prolyl isomerization phase, it folds in a two-step process, albeit very slowly (due to abundance of long-range interactions; ref. 9), through a relatively compact, native-like transition state. The loop we chose for the modifications (hereafter referred to as L4) connects between the second helix and the fourth β-strand of the protein (Fig. 1) and possesses multiple internal and external contacts (refs. 3 and 4 and our own contact analysis). The latter contacts are formed predominantly with residues located in the first loop of the protein (L1), which runs along L4 and is involved in the binding of the phosphate group of the substrate (4,(11)(12)(13)(14)(15).Characterizing the properties of hmAcP mutants carrying deletions or insertions in L4 we found that the thermodynamic stability of mutants in which the loop was shortened is increased to an extent significantly larger than that predicted by polymer models for loop closure entropy. The increased stability is predominantly due to a decrease in the unfolding rate and is attained despite the fact that sho...

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and pliant interaction network between cargo and FG repeats.

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability.

Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for alpha/beta proteins. Unfolding is initiated at the C-terminal strand (beta(T)) that lies at one edge of the beta-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with beta(T) of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.

In dicots, the key developmental process by which immature plastids differentiate into photosynthetically competent chloroplasts commences in the shoot apical meristem (SAM), within the shoot apex. Using laser-capture microdissection and single-cell RNA sequencing methodology, we studied the changes in the transcriptome along the chloroplast developmental pathway in the shoot apex of tomato seedlings. The analysis revealed the presence of transcripts for different chloroplast functions already in the stem cell-containing region of the SAM. Thereafter, an en masse up-regulation of genes encoding for various proteins occurs, including chloroplast ribosomal proteins and proteins involved in photosynthesis, photoprotection and detoxification of reactive oxygen species. The results highlight transcriptional events that operate during chloroplast biogenesis, leading to the rapid establishment of photosynthetic competence.