Showing papers in "Protein Science in 2017"

Diving into traversable wormholes

Abstract: We study various aspects of wormholes that are made traversable by an interaction beween the two asymptotic boundaries. We concentrate on the case of nearly-AdS2 gravity and discuss a very simple mechanical picture for the gravitational dynamics. We derive a formula for the two sided correlators that includes the effect of gravitational backreaction, which limits the amount of information we can send through the wormhole. We emphasize that the process can be viewed as a teleportation protocol where the teleportee feels nothing special as he/she goes through the wormhole. We discuss some applications to the cloning paradox for old black holes. We point out that the same formula we derived for AdS2 gravity is also valid for the simple SYK quantum mechanical theory, around the thermofield double state. We present a heuristic picture for this phenomenon in terms of an operator growth model. Finally, we show that a similar effect is present in a completely classical chaotic system with a large number of degrees of freedom.

Spheres, Generalised Parallelisability and Consistent Truncations

Abstract: We show that generalised geometry gives a unified description of maximally supersymmetric consistent truncations of ten- and eleven-dimensional supergravity. In all cases the reduction manifold admits a “generalised parallelisation” with a frame algebra with constant coefficients. The consistent truncation then arises as a generalised version of a conventional Scherk–Schwarz reduction with the frame algebra encoding the embedding tensor of the reduced theory. The key new result is that all round-sphere Sd geometries admit such generalised parallelisations with an SO(d+1) frame algebra. Thus we show that the remarkable consistent truncations on S3, S4, S5 and S7 are in fact simply generalised Scherk–Schwarz reductions. This description leads directly to the standard non-linear scalar-field ansatze and as an application we give the full scalar-field ansatz for the type IIB truncation on S5.

L∞ algebras and field theory

Abstract: We review and develop the general properties of L∞ algebras focusing on the gauge structure of the associated field theories. Motivated by the L∞ homotopy Lie algebra of closed string field theory and the work of Roytenberg and Weinstein describing the Courant bracket in this language we investigate the L∞ structure of general gauge invariant perturbative field theories. We sketch such formulations for non-abelian gauge theories, Einstein gravity, and for double field theory. We find that there is an L∞ algebra for the gauge structure and a larger one for the full interacting field theory. Theories where the gauge structure is a strict Lie algebra often require the full L∞ algebra for the interacting theory. The analysis suggests that L∞ algebras provide a classification of perturbative gauge invariant classical field theories.

Monobodies and other synthetic binding proteins for expanding protein science.

Abstract: Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

Comparison of force fields for Alzheimer's A β42: A case study for intrinsically disordered proteins.

Abstract: Intrinsically disordered proteins are essential for biological processes such as cell signalling, but are also associated to devastating diseases including Alzheimer's disease, Parkinson's disease or type II diabetes. Because of their lack of a stable three-dimensional structure, molecular dynamics simulations are often used to obtain atomistic details that cannot be observed experimentally. The applicability of molecular dynamics simulations depends on the accuracy of the force field chosen to represent the underlying free energy surface of the system. Here, we use replica exchange molecular dynamics simulations to test five modern force fields, OPLS, AMBER99SB, AMBER99SB*ILDN, AMBER99SBILDN-NMR and CHARMM22*, in their ability to model Aβ42 , an intrinsically disordered peptide associated with Alzheimer's disease, and compare our results to nuclear magnetic resonance (NMR) experimental data. We observe that all force fields except AMBER99SBILDN-NMR successfully reproduce local NMR observables, with CHARMM22* being slightly better than the other force fields.

A cell is more than the sum of its (dilute) parts: A brief history of quinary structure.

Abstract: Most knowledge of protein structure and function is derived from experiments performed with purified protein resuspended in dilute, buffered solutions. However, proteins function in the crowded, complex cellular environment. Although the first four levels of protein structure provide important information, a complete understanding requires consideration of quinary structure. Quinary structure comprises the transient interactions between macromolecules that provides organization and compartmentalization inside cells. We review the history of quinary structure in the context of several metabolic pathways, and the technological advances that have yielded recent insight into protein behavior in living cells. The evidence demonstrates that protein behavior in isolated solutions deviates from behavior in the physiological environment.

Amyloidogenesis of Tau protein.

Abstract: The role of microtubule-associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post-translational modifications, or interactions with polyanionic molecules and aggregation-prone proteins/peptides. The self-assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate-limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates ("seeds"). Accordingly, Tau aggregates released by tauopathy-affected neurons can spread the neurodegenerative process in the brain through a prion-like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains-structurally diverse self-propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion-like paradigm.

Modulating the wnt signaling pathway with small molecules

Abstract: Wnt signaling is a critical component during embryonic development and also plays an important role in regulating adult tissue homeostasis. Abnormal activation of Wnt signaling has been implicated in many cancers, while reduced activity of Wnt signaling leads to poor wound healing and structural formations. Thus, extensive efforts have been focused on developing small molecules that have potential to either inhibit or activate the pathway, hoping these molecules can offer leads for novel approaches in treating different human diseases. Many small-molecule inhibitors specifically target various elements, such as Frizzled, Disheveled, Porcupine, or Tankyrase, within the Wnt signaling pathways. These small molecules not only have the potential to be further developed as therapeutic reagents, but they may also be used as chemical probes to dissect the underlying mechanism of the Wnt signaling pathways. Therefore, their respective mechanisms and effective dosages are highly pertinent. Aiming to provide an overview of those molecules in a concise, easy-to-use manner, we summarize and organize the current research on them so that it may be helpful for utilization in different studies.

How cryo-electron microscopy and X-ray crystallography complement each other.

Abstract: With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms.

Exotic RG Flows from Holography

Abstract: Holographic RG flows are studied in an Einstein-dilaton theory with a general potential. The superpotential formalism is utilized in order to characterize and classify all solutions that are associated with asymptotically AdS space-times. Such solutions correspond to holographic RG flows and are characterized by their holographic β-functions. Novel solutions are found that have exotic properties from a RG point-of view. Some have β-functions that are defined patch-wise and lead to flows where the β-function changes sign without the flow stopping. Others describe flows that end in non-neighboring extrema in field space. Finally others describe regular flows between two minima of the potential and correspond holographically to flows driven by the VEV of an irrelevant operator in the UV CFT.

New Gaugings and Non‐Geometry

Abstract: We discuss the possible realisation in string/M theory of the recently discovered family of four-dimensional maximal SO(8) gauged supergravities, and of an analogous family of seven-dimensional half-maximal SO(4) gauged supergravities. We first prove a no-go theorem that neither class of gaugings can be realised via a compactification that is locally described by ten- or eleven-dimensional supergravity. In the language of Double Field Theory and its M theory analogue, this implies that the section condition must be violated. Introducing the minimal number of additional coordinates possible, we then show that the standard S3 and S7 compactifications of ten- and eleven-dimensional supergravity admit a new class of section-violating generalised frames with a generalised Lie derivative algebra that reproduces the embedding tensor of the SO(4) and SO(8) gaugings respectively. The physical meaning, if any, of these constructions is unclear. They highlight a number of the issues that arise when attempting to apply the formalism of Double Field Theory to non-toroidal backgrounds. Using a naive brane charge quantisation to determine the periodicities of the additional coordinates restricts the SO(4) gaugings to an infinite discrete set and excludes all the SO(8) gaugings other than the standard one.

Half‐Maximal Supersymmetry from Exceptional Field Theory

Abstract: We study D4-dimensional half-maximal flux backgrounds using exceptional field theory. We define the relevant generalised structures and also find the integrability conditions which give warped half-maximal Minkowski(D) and AdS(D) vacua. We then show how to obtain consistent truncations of type II / 11-dimensional SUGRA which break half the supersymmetry. Such truncations can be defined on backgrounds admitting exceptional generalised SO (d-1-N) structures, where d=11-D, and N is the number of vector multiplets obtained in the lower-dimensional theory. Our procedure yields the most general embedding tensors satisfying the linear constraint of half-maximal gauged SUGRA. We use this to prove that all D4 half-maximal warped AdS(D) and Minkowski(D) vacua of type II / 11-dimensional SUGRA admit a consistent truncation keeping only the gravitational supermultiplet. We also show to obtain heterotic double field theory from exceptional field theory and comment on the M-theory / heterotic duality. In five dimensions, we find a new SO(5, N) double field theory with a (6+N)-dimensional extended space. Its section condition has one solution corresponding to 10-dimensional N=1 supergravity and another yielding six-dimensional N=(2,0) SUGRA.

Structural and functional diversity of EF-hand proteins: Evolutionary perspectives.

Abstract: We have classified 865 sequences of EF-hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF-lobe (pair of adjacent EF-hands). They have two or more EF-lobes; the relative positions of their EF-lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF-lobes of these groups indicate that the mutual positions of EF-lobes were established soon after divergence of an EF-lobe for each group and before the duplication and fusion of EF-lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF-lobe precursor, even if their conformations are similar to the canonical EF-hand. This is an example of convergent evolution.

On Inflation and de Sitter in Non-Geometric String Backgrounds

Abstract: We study the problem of obtaining de Sitter and inflationary vacua from dimensional reduction of double field theory (DFT) on nongeometric string backgrounds. In this context, we consider a new class of effective potentials that admit Minkowski and de Sitter minima. We then construct a simple model of chaotic inflation arising from T-fold backgrounds and we discuss the possibility of trans-Planckian field range from nongeometric monodromies as well as the conditions required to get slow roll.

Exceptional Calabi–Yau spaces: the geometry of N=2 backgrounds with flux

Abstract: In this paper we define the analogue of Calabi–Yau geometry for generic D=4, N=2 flux backgrounds in type II supergravity and M-theory. We show that solutions of the Killing spinor equations are in one-to-one correspondence with integrable, globally defined structures in E7(7)×R+ generalised geometry. Such “exceptional Calabi–Yau” geometries are determined by two generalised objects that parametrise hyper- and vector-multiplet degrees of freedom and generalise conventional complex, symplectic and hyper-Kahler geometries. The integrability conditions for both hyper- and vector-multiplet structures are given by the vanishing of moment maps for the “generalised diffeomorphism group” of diffeomorphisms combined with gauge transformations. We give a number of explicit examples and discuss the structure of the moduli spaces of solutions. We then extend our construction to D=5 and D=6 flux backgrounds preserving eight supercharges, where similar structures appear, and finally discuss the analogous structures in O(d,d)×R+ generalised geometry.

Building collagen IV smart scaffolds on the outside of cells.

Abstract: Collagen IV scaffolds assemble through an intricate pathway that begins intracellularly and is completed extracellularly. Multiple intracellular enzymes act in concert to assemble collagen IV protomers, the building blocks of collagen IV scaffolds. After being secreted from cells, protomers are activated to initiate oligomerization, forming insoluble networks that are structurally reinforced with covalent crosslinks. Within these networks, embedded binding sites along the length of the protomer lead to the "decoration" of collagen IV triple helix with numerous functional molecules. We refer to these networks as "smart" scaffolds, which as a component of the basement membrane enable the development and function of multicellular tissues in all animal phyla. In this review, we present key molecular mechanisms that drive the assembly of collagen IV smart scaffolds.

Breaking up and making up: The secret life of the vacuolar H+-ATPase

Abstract: The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Thermodynamic deficiencies of some simple Lindblad operators

Abstract: Master equations of Lindblad type have attained prominent status in the fields of quantum optics and quantum information since they are guaranteed to satisfy fundamental notions of quantum dynamics such as complete positivity. When Lindblad operators are used to describe thermal reservoirs in contact with an open quantum system, the fundamental laws of thermodynamics and the fluctuation-dissipation theorem provide additional mandatory criteria. We show several examples of innocent-looking Lindblad operators which have questionable properties in this regard. Compatibility criteria between Hamiltonian and Lindblad terms as well as consequences of their violation are discussed. An alternative stochastic approach to dissipative quantum dynamics is outlined and illustrated through a harmonic-chain model for which the approach of local Lindblad operators fails.

Advances in quantum control of three-level superconducting circuit architectures

Abstract: Advanced control in Lambda (Λ) scheme of a solid state architecture of artificial atoms and quantized modes would allow the translation to the solid-state realm of a whole class of phenomena from quantum optics, thus exploiting new physics emerging in larger integrated quantum networks and for stronger couplings. However control solid-state devices has constraints coming from selection rules, due to symmetries which on the other hand yield protection from decoherence, and from design issues, for instance that coupling to microwave cavities is not directly switchable. We present two new schemes for the Λ-STIRAP control problem with the constraint of one or two classical driving fields being always-on. We show how these protocols are converted to apply to circuit-QED architectures. We finally illustrate an application to coherent spectroscopy of the so called ultrastrong atom-cavity coupling regime.

Molecular stretching modulates mechanosensing pathways.

Abstract: For individual cells in tissues to create the diverse forms of biological organisms, it is necessary that they must reliably sense and generate the correct forces over the correct distances and directions. There is considerable evidence that the mechanical aspects of the cellular microenvironment provide critical physical parameters to be sensed. How proteins sense forces and cellular geometry to create the correct morphology is not understood in detail but protein unfolding appears to be a major component in force and displacement sensing. Thus, the crystallographic structure of a protein domain provides only a starting point to then analyze what will be the effects of physiological forces through domain unfolding or catch-bond formation. In this review, we will discuss the recent studies of cytoskeletal and adhesion proteins that describe protein domain dynamics. Forces applied to proteins can activate or inhibit enzymes, increase or decrease protein-protein interactions, activate or inhibit protein substrates, induce catch bonds and regulate interactions with membranes or nucleic acids. Further, the dynamics of stretch-relaxation can average forces or movements to reliably regulate morphogenic movements. In the few cases where single molecule mechanics are studied under physiological conditions such as titin and talin, there are rapid cycles of stretch-relaxation that produce mechanosensing signals. Fortunately, the development of new single molecule and super-resolution imaging methods enable the analysis of single molecule mechanics in physiologically relevant conditions. Thus, we feel that stereotypical changes in cell and tissue shape involve mechanosensing that can be analyzed at the nanometer level to determine the molecular mechanisms involved.

S)Pinning down protein interactions by NMR

Abstract: Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions. These types of analyses determine the amounts and proportions of individual constituents that participate in a reaction as well as their rates of reactions and their thermodynamics. Although many different methods are available, there is currently no single method able to quantitatively capture and describe all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates, and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background and illustrate simple protein-ligand interactions as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction.

Effect of the acylation of TEAD4 on its interaction with co-activators YAP and TAZ

Abstract: The Hippo pathway is deregulated in various cancers, and the discovery of molecules that modulate this pathway may open new therapeutic avenues in oncology. TEA/ATTS domain (TEAD) transcription factors are the most distal elements of the Hippo pathway and their transcriptional activity is regulated by the Yes-associated protein (YAP). Amongst the various possibilities for targeting this pathway, inhibition of the YAP:TEAD interaction is an attractive strategy. It has been shown recently that TEAD proteins are covalently linked via a conserved cysteine to a fatty acid molecule (palmitate) that binds to a deep hydrophobic cavity present in these proteins. This acylation of TEAD seems to be required for efficient binding to YAP, and understanding how it modulates the YAP:TEAD interaction may provide useful information on the regulation of TEAD function. In this report we have studied the effect of TEAD4 acylation on its interaction with YAP and the other co-activator transcriptional co-activator with PDZ-binding motif (TAZ). We show in our biochemical and cellular assays that YAP and TAZ bind in a similar manner to acylated and non-acylated TEAD4. This indicates that TEAD4 acylation is not a prerequisite for its interaction with YAP or TAZ. However, we observed that TEAD4 acylation significantly enhances its stability, suggesting that it may help this transcription factor to acquire and/or maintain its active conformation.

What is the magnetic Weak Gravity Conjecture for axions

Abstract: The electric Weak Gravity Conjecture demands that axions with large decay constant f couple to light instantons. The resulting large instantonic corrections pose problems for natural inflation. We explore an alternative argument based on the magnetic Weak Gravity Conjecture for axions, which we try to make more precise. Roughly speaking, it demands that the minimally charged string coupled to the dual 2-form-field exists in the effective theory. Most naively, such large-f strings curve space too much to exist as static solutions, thus ruling out large-f axions. More conservatively, one might allow non-static string solutions to play the role of the required charged objects. In this case, topological inflation would save the superplanckian axion. Furthermore, a large-f axion may appear in the low-energy effective theory based on two subplanckian axions in the UV. The resulting effective string is a composite object built from several elementary strings and domain walls. It may or may not satisfy the magnetic Weak Gravity Conjecture depending on how strictly the latter is interpreted and on the cosmological dynamics of this composite object, which remain to be fully understood. Finally, we recall that large-field brane inflation is naively possible in the codimension-one case. We show how string-theoretic back-reaction closes this apparent loophole of large-f (non-periodic) pseudo-axions.

Targeting human SET1/MLL family of proteins.

Abstract: The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.

On the interpretation of electron microscopic maps of biological macromolecules

Abstract: The images of flash-frozen biological macromolecules produced by cryo-electron microscopy (EM) can be used to generate accurate, three-dimensional, electric potential maps for these molecules that resemble X-ray-derived electron density maps. However, unlike electron density maps, electric potential maps can include negative features that might for example represent the negatively charged, backbone phosphate groups of nucleic acids or protein carboxylate side chains, which can complicate their interpretation. This study examines the images of groups that include charged atoms that appear in recently-published, high-resolution EM potential maps of the ribosome and β-galactosidase. Comparisons of simulated maps of these same groups with their experimental counterparts highlight the impact that charge has on the appearance of electric potential maps.

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Abstract: Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2ʹ,3ʹ-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2ʹ,3ʹ-cGAMP formation, and interestingly, describe a catalytic mechanism where 2ʹ,3ʹ-cGAMP may be a minor product of cGAS compared to linear nucleotides. This article is protected by copyright. All rights reserved.

Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Abstract: Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

The IDL of E-coli SSB links ssDNA and protein binding by mediating protein-protein interactions

Abstract: The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.

Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

Abstract: Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

The ribosome destabilizes native and non-native structures in a nascent multidomain protein.

Abstract: Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multi-domain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multi-domain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multi-domain proteins. This article is protected by copyright. All rights reserved.