Unfolding Landscape Research Articles

Global Disordering in Stereo-Specific Protein Association

Protein-protein recognition is of fundamental importance for a myriad of biological processes and is ultimately a prerequisite for life as we know it. There exist several established mechanisms that promote formation of stereo-specific protein complexes. Many of these mechanism involve conformational changes of one or both proteins in dimeric assemblies as observed in “conformational selection” and “coupled folding and binding” scenarios. In “coupled folding and binding” events, at least one of the proteins undergoes a global ordering event. By using an integrated computational and experimental approach we have discovered that also global disordering can be a productive route for formation of a stereo-specific protein complex. This mechanism was observed for the chaperone binding domain of the Yersinia effector protein YopH upon binding to its specific chaperone SycH. These two proteins are crucial for type III secretion system mediated infectivity by Yersinia and several other gram negative pathogens. NMR relaxation dispersion experiments demonstrated that the otherwise well folded YopH protein dynamically samples an expanded high-energy state that corresponds to the SycH binding competent conformation. A structure of the protein complex determined from a hybrid SAXS and computational approach revealed that YopH wraps around SycH in a horse shoe like conformation. The binding model was validated by site specific YopH mutations that promoted the disordering event and at the same time displayed improved binding affinity towards SycH. Taken together the data illustrates a tight coupling between a proteins unfolding and functional free energy landscapes and add valuable mechanistic insight into protein-protein recognition.

Structural and Mechanistic Insights into the Copper-Modulated Unfolding Pathways of Azurin

Characterization of energy landscape of metalloproteins is challenging because of the subtle changes imparted by metal binding on the stability and conformational flexibility of proteins. Here we present copper-binding induced changes in the mechanical unfolding pathways of azurin, a β-barrel protein with type-I copper center, through a joint experimental-computational study. Single-molecule force spectroscopy experiments reveal an intermediate along the unfolding pathway of apo-azurin in half of population. Steered molecular dynamics simulations attribute the native and intermediate unfolding transition states (TSs) to the rupture of interactions between the pairs of β-strands, 2B-8 and 4-7, respectively. We show that the copper-binding does not change the first TS of azurin and its mechanical properties, but influences the second TS. The rupture of 4-7 β-strand pair is delayed in holo-azurin because of constraints imposed by the copper coordination sphere and occurs after the second TS. Our experimental and computational approach extracts unprecedented details along the unfolding landscape for apo- and holo- forms of azurin.

Ripping RNA by Force Using Gaussian Network Models

Using force as a probe to map the folding landscapes of RNA molecules has become a reality thanks to major advances in single molecule pulling experiments. Although the unfolding pathways under tension are complicated to predict, studies in the context of proteins have shown that topology is the major determinant of the unfolding landscapes. By building on this finding we study the responses of RNA molecules to force by adapting Gaussian network model (GNM) that represents RNAs using a bead-spring network with isotropic interactions. Cross-correlation matrices of residue fluctuations, which are analytically calculated using GNM even upon application of mechanical force, show distinct allosteric communication as RNAs rupture. The model is used to calculate the force-extension curves at full thermodynamic equilibrium, and the corresponding unfolding pathways of four RNA molecules subject to a quasi-statically increased force. Our study finds that the analysis using GNM captures qualitatively the unfolding pathway of T. ribozyme elucidated by the optical tweezers measurement. However, the simple model cannot capture features, such as bifurcation in the unfolding pathways or the ion effects, in the forced-unfolding of RNAs.

What Kind of Urban? A Case Study of Kullu, A Small Indian Town

This article examines the kind of urbanisation that is emerging in Kullu, a small but quickly growing North Indian mountain town. It does this both within the context of the town itself, but also by studying its relationship to nearby villages that are increasingly a part of the town’s remit as it emerges as a new metropolitan region. What the article seeks to highlight is that despite rising incomes, urban and spatial expansion and in-migration, many of the markers of new urban growth commonly found in other metropolitan Indian cities are absent in Kullu. These include increasing corporate real estate development, middle class and public consumptive cultures, neoliberal experiments with governance and rising urban inequality. The article locates the area’s tepid response to the templated ‘new’ Indian urban in a continuing orientation and attunement of Kullu residents to the high alpine Himalayan landscape within which the town is located and by which it is hemmed wherein belonging and dwelling are predicated on a continuing attentiveness to the unfolding landscape, and on rurality, in which many urban residents continue to have a stake. This, even as relatively widespread land distribution among those considered ‘local’ ensures a relatively well-distributed and languid prosperity in the area as a whole. In this context, the article argues for the rethinking of singular definitions of urbanisation and of the relationship between urban and rural areas, particularly in light of dominant metropolitan experiences, and to consider the alternative forms of urban life that Kullu presents. Simultaneously, however, it discusses new and emerging fault lines that threaten Kullu’s existing rural–urban compact in the concluding section, namely new youth cultures and the building practices of locals themselves.

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.

Civic open data at a crossroads: Dominant models and current challenges

As open data becomes more widely provided by government, it is important to ask questions about the future possibilities and forms that government open data may take. We present four models of open data as they relate to changing relations between citizens and government. These models include; a status quo ‘data over the wall’ form of government data publishing, a form of ‘code exchange’, with government acting as an open data activist, open data as a civic issue tracker, and participatory open data. These models represent multiple end points that can be currently viewed from the unfolding landscape of government open data. We position open data at a crossroads, with significant concerns of the conflicting motivations driving open data, the shifting role of government as a service provider, and the fragile nature of open data within the government space. We emphasize that the future of open data will be driven by the negotiation of the ethical-economic tension that exists between provisioning governments, citizens, and private sector data users.

Specific Binding of the Nucleocapsid Protein Transforms the Folding Landscape of the HIV-1 TAR RNA Hairpin

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC destabilizes nucleic acids, including the transactivation response (TAR) hairpin to facilitate their structural re-arrangement into the lowest free energy conformations. By combining single molecule optical tweezers measurements with mfold-based free energy calculations of the unfolding landscape, we determine the equilibrium TAR stability and characterize the unfolding transition state in both the presence and absence of NC. Our results show that protein binding occurs specifically at guanines that border structural defects in the upper part of TAR hairpin. This results in preferential destabilization by NC of this part of TAR, thereby shifting the transition state closer to the bottom of the TAR stem, leading to a shorter critical unfolding length. Thus, NC facilitates TAR RNA annealing to its complementary DNA hairpin while having little effect on the stability of the final annealed duplexes. These results provide the first direct measurement of alterations in an RNA folding landscape that are induced by protein-RNA interactions, and are a novel case study of the effect of a protein that specifically destabilizes particular elements of the nucleic acid secondary structure.

Single-shot NMR measurement of protein unfolding landscapes

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine—Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is 15N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein β2-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.

Determining the Energy Landscape of Proteins by a Fast Isotope Exchange NMR Approach

We present a new and efficient NMR method, BLUU-Tramp (Biophysics Laboratory University of Udine temperature ramp), for the collection of hydrogen-deuterium exchange experiments as a function of time and temperature for small and medium-sized proteins. Exchange rates can be determined to extract the underlying thermodynamic equilibrium or kinetic parameters by sampling hundreds of points over a virtually continuous temperature ramp. Data are acquired in a single experimental session that lasts some 20-60 h, depending on the thermal stability of the protein. Subsequent analysis provides a complete thermodynamic description of the protein energy landscape. The global thermal unfolding process and the partial or local structure opening events can be fully determined at the single-residue resolution level. The proposed approach is shown to work successfully with the amyloidogenic protein β(2)-microglobulin. With (15)N-labeling, the unfolding landscape of a protein can also be studied in the presence of other unlabeled proteins and, in general, with ligands or cosolutes or in physiological environments.

Car talk: Integrating texts, bodies, and changing landscapes

Making use of videotaped recordings of interaction in cars filmed as middle class families pursue their daily activities, we examine some of the ways in which talk while driving includes as parts of its intrinsic organization ongoing attention to phenomena beyond the stream of speech. Important consideration is given to issues posed by the task of driving while talking about a seeable field in either the unfolding landscape or a textual artifact within the car itself. Of particular interest to our analysis is how such phenomena are attended to, collaboratively recognized, and incorporated into the ongoing organization of talk. This process involves making use of a range of resources including deictics, perceptual directives, address terms, pointings (C. Goodwin 2003), etc., to locate for others these phenomena, as well as forms of stance display that inform how the speaker aligns towards the event. Through their gaze direction, questions, and displays of understandings recipients can display their response to a noticing or reporting.

The unfolding treatment landscape for men with castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is a fatal disease in virtually all patients. Docetaxel chemotherapy became the standard front-line agent based on the results of the TAX327 trial in 2004, with a survival advantage of 3 months achieved over mitoxantrone. Over the past few years, an improved understanding of the molecular biology of castration-resistance has resulted in expansion of the treatment armamentarium for advanced prostate cancer with the emergence of novel androgen receptor-directed therapies, cytotoxic chemotherapies, as well as immunotherapies. Four different agents have very recently gained approval by the U.S. Food and Drug Administration for the treatment of CRPC and this review will summarize the development, mechanism of action, and safety and efficacy of these agents as demonstrated in preclinical as well as clinical studies.

Mechanically Unfolding Protein L Using a Laser-Feedback-Controlled Cantilever

Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique—constant-deflection AFM—in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.

On mapping and its afterlife: unfolding landscapes in northwestern North America

This paper contributes to recent postcolonial debates about the cartography of European expansion and its implications for constructing North American landscapes. It does so by moving beyond a pure deconstructionist critique of mapping by drawing on range of theoretical literature that encourages a view of cartography as being inextricably tied to the social relations of production and consumption. In particular, it emphasizes the point that maps have social lives not necessarily different in kind from other forms of material culture. Drawing on the cartographic creation of the northwest, the argument develops through examining the social and material dimensions of map making and its afterlife. The paper suggests, therefore, that the ‘power of maps’ had varying and often contradictory outcomes.

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5'-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt-Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT-TTR and L55P-TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P-TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single alpha-helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation-prone conformations characterized by full displacement of strands C and D from the main beta-sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H-bond network and consequent destabilization of the CBEF beta-sheet of the beta-sandwich; (v) WT forms aggregation-compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P-TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.

Real-time NMR Kinetic Studies Provide Global and Residue-specific Information on the Non-cooperative Unfolding of the β-Trefoil Protein, Interleukin-1β

The interleukin-1β (IL-1β) structural motif is a β-trefoil super fold created by six two-stranded β-hairpins. Turns are thus particularly important in creating the topology and the arrangement of β-strands in this structural motif. In contrast to the signals observed in optical studies, real-time NMR kinetic investigations of the denaturant-induced unfolding of interleukin-1β provide direct, global, and residue-specific information on the structural nature of the unfolding reaction. Heterogeneity in the individual amino acid residue kinetics reveals a rugged unfolding landscape. The relative kinetic stability of native-like turns supports low temperature molecular dynamics predictions of turn-controlled unfolding.

Simulations of Human Lysozyme: Probing the Conformations Triggering Amyloidosis

A natural mutant of human lysozyme, D67H, causes hereditary systemic nonneuropathic amyloidosis, which can be fatal. In this disease, insoluble β-stranded fibrils (amyloids) are found in tissues stemming from the aggregation of partially folded intermediates of the mutant. In this study, we specifically compare the conformation and properties of the structures adopted from the induced unfolding, at elevated temperature, using molecular dynamics. To increase the sampling of the unfolding conformational landscape, three 5 ns trajectories are performed for each of the wild-type and mutant D67H proteins resulting in a total of 30 ns simulation. Our results show that the mutant unfolds slightly faster than the wild-type with both wild-type and mutant proteins losing most of their native secondary structure within the first 2 ns. They both develop random transient β-strands across the whole polypeptide chain. Clustering analysis of all the conformations shows that a high population of the mutant protein conformations have a distorted β-domain. This is consistent with experimental results suggesting that this region is pivotal in the formation of conformations prone to act as “seeds” for amyloid fiber formation.