Ruti Kapon

Direct measurement of protein energy landscape roughness

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 ε for a complex consisting of the small GTPase Ran and the nuclear transport receptor importin‐β. The results gave ε>5kBT, indicating a bumpy energy surface, which is consistent with the ability of importin‐β to accommodate multiple conformations and to interact with different, structurally distinct ligands.

Passive and facilitated transport in nuclear pore complexes is largely uncoupled.

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.

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

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.

Scanning force microscopy in the applied biological sciences

Fifteen years after its invention, the scanning force microscope (SFM) is rooted deep in the biological sciences. Here we discuss the use of SFM in biotechnology and biomedical research. The spectrum of applications reviewed includes imaging, force spectroscopy and mapping, as well as sensor applications. It is our hope that this review will be useful for researchers considering the use of SFM in their studies but are uncertain about its scope of capabilities. For the benefit of readers unfamiliar with SFM technology, the fundamentals of SFM imaging and force measurement are also briefly introduced.

Stabilization of a protein conferred by an increase in folded state entropy

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.

A possible mechanism for self-coordination of bidirectional traffic across nuclear pores

Nuclear pore complexes are constantly confronted by large fluxes of macromolecules and macromolecular complexes that need to get into and out of the nucleus. Such bidirectional traffic occurring in a narrow channel can easily lead to jamming. How then is passage between the nucleus and cytoplasm maintained under the varying conditions that arise during the lifetime of the cell? Here, we address this question using computer simulations in which the behaviour of the ensemble of transporting cargoes is analysed under different conditions. We suggest that traffic can exist in two distinct modes, depending on the concentration of cargoes and dissociation rates of the transport receptor-cargo complexes from the pores. In one mode, which prevails when dissociation is quick and cargo concentration is low, transport in either direction proceeds uninterrupted by transport in the other direction. The result is that the overall traffic direction fluctuates rapidly and unsystematically between import and export. Remarkably, when cargo concentrations are high and disassociation is slow, another mode takes over in which traffic proceeds in one direction for a certain extent of time, after which it flips direction for another period. The switch between this, more regulated, mode of transport and the other, quickly fluctuating state, does not require an active gating mechanism but rather occurs spontaneously through the dynamics of the transported particles themselves. The determining factor for the behaviour of traffic is found to be the exit rate from the pore channel, which is directly related to the activity of the Ran system that controls the loading and release of cargo in the appropriate cellular compartment.

Permeating the nuclear pore complex

The extensive and multifaceted traffic between nucleus and cytoplasm is handled by a single type of macromolecular assembly called the nuclear pore complex (NPC). While being readily accessible to ions and metabolites, the NPC imposes stringent selectivity on the passage of proteins and RNA, tightly regulating their traffic between the two major cellular compartments. Here we discuss how shuttling carriers, which mediate the transport of macromolecules through NPCs, cross its permeability barrier. We also discuss the co-existence of receptor-mediated macromolecular transport with the passive diffusion of small molecules in the context of the various models suggested for the permeability barrier of the NPC. Finally, we speculate on how nuclear transport receptors negotiate the dependence of their NPC-permeating abilities on hydrophobic interactions with the necessity of avoiding these promiscuous interactions in the cytoplasm and nucleus.

Commuting within the cell—mind the GAPs. Workshop on Systems Dynamics of Intracellular Communication:Overcoming Distance in Signalling Networks

The EMBO/FEBS/ISF Workshop on Systems Dynamics of Intracellular Communication: Overcoming Distance in Signalling Networks took place between 18 and 22 March 2007, in the hills of Jerusalem, Israel, and was organized by M. Fainzilber, B. Kholodenko and G. Schiavo. ![][1] The first EMBO/FEBS/ISF workshop on Systems Dynamics of Intracellular Communication: Overcoming Distance in Signalling Networks was held between the 18 and 22 of March 2007, in Maale Hachamisha, a Kibutz in the hills of Jerusalem, Israel. It brought together scientists from diverse fields of research to discuss the latest developments and theories about how information originating in the extracellular environment travels to the genome to achieve a cellular homeostatic response. Iteration between mathematical modelling and experimentation is essential to the understanding of such complex processes, but this is usually easier said than done; however, at this meeting, communication between theoreticians and experimentalists was outstanding and led to lively discussions, and to what all participants agreed was a very successful first workshop. It was decided, after a vote held at the end of the meeting, to try to organize a second meeting in 2009 at the same place; perhaps owing to the desire of some participants to follow up on the belly dancing lessons provided after the gala dinner. Here, we summarize the main topics discussed at the meeting. Sadly, Reinhart Heinrich, who was scheduled to speak at the workshop, passed away in October 2006. In his memory, B. Kholodenko (Philadelphia, PA, USA) opened the meeting with a description of Professor Heinrichs many contributions as one of the founders of systems biology. Kholodenko followed on this theme by introducing the role of feedback loops as control mechanisms of many cellular processes. A central role for feedback loops and their modulators—most notably GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs)—in controlling … [1]: /embed/graphic-1.gif

Probing the Energy Landscape of Protein-Binding Reactions by Dynamic Force Spectroscopy

This chapter describes how the energy landscape that underlies protein-binding reactions can be revealed using dynamic force spectroscopy. The chapter begins with a detailed description of methodologies used and requirements of the experimental system, including tip and surface materials and their functionalization strategies. The next few sections discuss the fundamentals of measuring forces using the atomic force microscope, and the basics of performing force spectroscopy measurements from a practical point of view. Next, it presents an extensive account of methods for data analysis and current theoretical treatments. The remainder of the chapter illustrates the power of this methodology by several examples in which the location of energy barriers in a binding reaction pathway and their load-dependent dynamics are measured, the overall scale of roughness of the underlying energy surface is extracted, and alternative modes of protein activation are distinguished. Biological insight gained from these data is discussed. The intent is to provide the necessary theoretical and practical knowledge to begin force spectroscopy measurements on protein interactions.

Molecular Recognition Force Microscopy: From Molecular Bonds to Complex Energy Landscapes

Atomic force microscopy (AFM), developed in the late eighties to explore atomic details on hard material surfaces, has evolved to an imaging method capable of achieving fine structural details on biological samples. Its particular advantage in biology is that the measurements can be carried out in aqueous and physiological environment, which opens the possibility to study the dynamics of biological processes in vivo. The additional potential of the AFM to measure ultra-low forces at high lateral resolution has paved the way for measuring inter- and intra-molecular forces of bio-molecules on the single molecule level. Molecular recognition studies using AFM open the possibility to detect specific ligand–receptor interaction forces and to observe molecular recognition of a single ligand–receptor pair. Applications include biotin–avidin, antibody–antigen, NTA nitrilotriacetate–hexahistidine 6, and cellular proteins, either isolated or in cell membranes. The general strategy is to bind ligands to AFM tips and receptors to probe surfaces (or vice versa), respectively. In a force–distance cycle, the tip is first approached towards the surface whereupon a single receptor–ligand complex is formed, due to the specific ligand receptor recognition. During subsequent tip–surface retraction a temporarily increasing force is exerted to the ligand–receptor connection thus reducing its lifetime until the interaction bond breaks at a critical force (unbinding force). Such experiments allow for estimation of affinity, rate constants, and structural data of the binding pocket. Comparing them with values obtained from ensemble-average techniques and binding energies is of particular interest. The dependences of unbinding force on the rate of load increase exerted to the receptor–ligand bond reveal details of the molecular dynamics of the recognition process and energy landscapes. Similar experimental strategies were also used for studying intra-molecular force properties of polymers and unfolding–refolding kinetics of filamentous proteins. Recognition imaging, developed by combing dynamic force microscopy with force spectroscopy, allows for localization of receptor sites on surfaces with nanometer positional accuracy.