The energy landscape of proteins is thought to have an intricate, corrugated structure. Such roughness should have important consequences on the folding and binding kinetics of proteins, as well as on their equilibrium fluctuations. So far, no direct measurement of protein energy landscape roughness has been made. Here, we combined a recent theory with single-molecule dynamic force spectroscopy experiments to extract the overall energy scale of roughness e for a complex consisting of the small GTPase Ran and the nuclear transport receptor importin-b. The results gave e45k B T, indicating a bumpy energy surface, which is consistent with the ability of importin-b to accommodate multiple conformations and to interact with different, structurally distinct ligands.

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...

Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.Eukaryotic cell nuclei are separated from the cytoplasm by a double lipid bilayer system known as the nuclear envelope (NE).2 Exchange of material between the two compartments proceeds through nuclear pore complexes (NPCs), large protein assemblies that span the NE and provide the sole medium for exchange. The vertebrate NPC has a molecular mass of ϳ125 MDa (1) and is made up of ϳ30 different proteins, called nucleoporins, most of which are present in multiples of eight (2, 3). The core of the NPC consists of a symmetrical framework, measuring ϳ120 ϫ 90 nm, which is made of two coaxial rings sandwiching a wheel-like array of eight spoke-shaped domains. The spoke-ring assembly encircles the central pore channel, which resembles an hourglass 45-50 nm wide at its waist (4 -7). In addition to the central framework, NPCs contain peripheral structures, which are anchored to the ring moieties of the spoke-ring assembly and serve as docking sites for nuclear transport receptors and effectors. These structures include eight short (ϳ50 nm) filaments that protrude toward the cytoplasm and a massive, fish trap-like structure, termed the nuclear basket, which extends into the nucleus (4 -11). Yeast NPCs have an overall similar architecture but are smaller (3,(12)(13)(14).Transport across the NPC has been reviewed in detail (15-22) and can be divided into two modes. Small molecules, such as ions, metabolites, and intermediate-sized macromolecules, can pass unassisted by diffusion which becomes increasingly restricted as the particle approaches a size limit of ϳ10 nm in diameter (23,24). In contrast, proteins, RNAs, and their complexes are ushered selectively by dedicated soluble transport receptors, which recognize specific import (NLS) or nuclear export signal (NES) peptides displayed by the cargo. In most cases, assembly and disassembly of transport complexes in the appropriate cellular compartment are coordinated by the small GTPase Ran, which performs these activities by binding to transport receptors in its GTP-bound form. Facilitated translocation is often coupled to an input of metabolic energy that permits transport ...

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.

Plant photosynthetic (thylakoid) membranes are organized into complex networks that are differentiated into 2 distinct morphological and functional domains called grana and stroma lamellae. How the 2 domains join to form a continuous lamellar system has been the subject of numerous studies since the mid-1950s. Using different electron tomography techniques, we found that the grana and stroma lamellae are connected by an array of pitch-balanced right- and left-handed helical membrane surfaces of different radii and pitch. Consistent with theoretical predictions, this arrangement is shown to minimize the surface and bending energies of the membranes. Related configurations were proposed to be present in the rough endoplasmic reticulum and in dense nuclear matter phases theorized to exist in neutron star crusts, where the right- and left-handed helical elements differ only in their handedness. Pitch-balanced helical elements of alternating handedness may thus constitute a fundamental geometry for the efficient packing of connected layers or sheets.