Kuhnian revolutions in neuroscience: the role of tool development

The terms “paradigm” and “paradigm shift” originated in “The Structure of Scientific Revolutions” by Thomas Kuhn. A paradigm can be defined as the generally accepted concepts and practices of a field, and a paradigm shift its replacement in a scientific revolution. A paradigm shift results from a crisis caused by anomalies in a paradigm that reduce its usefulness to a field. Claims of paradigm shifts and revolutions are made frequently in the neurosciences. In this article I will consider neuroscience paradigms, and the claim that new tools and techniques rather than crises have driven paradigm shifts. I will argue that tool development has played a minor role in neuroscience revolutions.     

A microbial sensor for discovering structural probes of protein misfolding and aggregation

In all cell types, protein homeostasis, or “proteostasis,” is maintained by sophisticated quality control networks that regulate protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. In one notable example, Escherichia coli employ a proteostasis system that determines whether substrates of the twin-arginine translocation (Tat) pathway are correctly folded and thus suitable for transport across the tightly sealed cytoplasmic membrane. Herein, we review growing evidence that the Tat translocase itself discriminates folded proteins from those that are misfolded and/or aggregated, preferentially exporting only the former. Genetic suppressors that inactivate this mechanism have recently been isolated and provide direct evidence for the participation of the Tat translocase in structural proofreading of its protein substrates. We also discuss how this discriminatory “folding sensor” has been exploited for the discovery of structural probes (e.g., sequence mutations, pharmacologic chaperones, intracellular antibodies) that modulate the folding and solubility of virtually any protein-of-interest, including those associated with aggregation diseases (e.g., α-synuclein, amyloid-β protein). Taken together, these studies highlight the utility of engineered bacteria for rapidly and inexpensively uncovering potent anti-aggregation factors.     

Exploration of compact protein conformations using the guided replication monte carlo method

We have studied the use of a new Monte Carlo (MC) chain generation algorithm, introduced by T. Garel and H. Orland[(1990) Journal of Physics A, Vol. 23, pp. L621–L626], for examining the thermodynamics of protein folding transitions and for generating candidate C^αbackbone structures as starting points for a de now protein structure paradigm. This algorithm, termed the guided replication Monte Carlo method, allows a rational approach to the introduction of known “native” folded characteristics as constraints in the chain generation process. We have shown this algorithm to be computationally very efficient in generating large ensembles of candidate C^αchains on the face centered cubic lattice, and illustrate its use by calculating a number of thermodynamic quantities related to protein folding characteristics. In particular, we have used this static MC algorithm to compute such temperature-dependent quantities as the ensemble mean energy, ensemble mean free energy, the heat capacity, and the mean-square radius of gyration. We also demonstrate the use of several simple “guide fields” for introducing protein-specific constraints into the ensemble generation process. Several extensions to our current model are suggested, and applications of the method to other folding related problems are discussed. © 1995 John Wiley & Sons, Inc.     

Perfect temperature for protein structure prediction and folding

We have investigated the influence of the “noise” of inevitable errors in energetic parameters on-protein structure prediction. Because of this noise, only a part of all the interactions operating in a protein chain can be taken into account, and therefore a search for the energy minimum becomes inadequate for protein structure prediction. One can rather rely on statistical mechanics: a calculation carried out at a temperature T_* somewhat below that of protein melting gives the best possible, though always approximate prediction. The early stages of protein folding also “take into account” only a part of all the interactions; consequently, the same temperature T_* is favorable for the self-organization of native-like intermediates in protein folding. © 1995 Wiley-Liss, Inc.     

Visibilities,invisibilities and twilight zones at the crime scene in Portugal

The Police are the first element of a chain of custody culminating in the court. The documents they produce mediate the understandings between the crime scene and the court. Based on the formal rules, the police give visibility to the narrative and assign legitimacy and credibility to their performance. However, the decision to turn certain aspects of the narrative visible, leaving others in a twilight zone may have repercussions in the production of a verdict. Based in a qualitative analysis of three Portuguese judicial proceedings in this paper I explore how the narrative constructed by the police, based on what they see and what is unseen, travels between subepistemic cultures. Looking at the visibilities, invisibilities and twilight zones, I will try to understand how police forces at the crime scene in Portugal construct their narratives through the use of biological traces and how these narratives are part of the construction of the evidence. I argue that in criminal investigations in Portugal, the production of a narrative with legal meaning in court can be conditioned by the co-existence of the epistemic subcultures of police work (different police forces at the crime scene) that have different knowledge, practices, understandings and ways of “seeing” the forensic evidence. The degree of technological enthusiasm that guides the performance of different police forces at the crime scene is reflected in the way they “see” the scene and in the sociocultural understandings that they produce. This technological enthusiasm and what I call here “selective professional vision” are mobilized at the crime scene and can impact the robustness and efficiency of the evidence presented in court.     

Connecting domination contracts

In Black Rights/White Wrongs, Charles Mills continues his critique of contemporary American political philosophy for ignoring issues of racial oppression, and in particular for ignoring the way that liberal social contracts rest on underlying domination contracts. In this commentary, I will discuss some of the new research inspired by Mills’ account of domination contracts, including recent accounts of the “capability contract” and the “species contract”, and explore how they relate to Mills’ own work on the “racial contract”. While this new research on diverse domination contracts confirms the richness of Mills’ analysis of the social contract tradition, it may also require some revisions to his own preferred vision of how we theorize racial justice.     

Solvation effects on the 31P-nmr chemical shifts and infrared spectra of phosphate diesters

The effects of organic solvents on the ³¹P-mr chemical shifts of various phosphate diesters have been investigated in water and mixed-organic solvent systems. The addition of organic solvents to cyclic phosphates and to diethyl phosphate causes large upfield shifts of the phosphorus resonance which are attributed to solvent-induced changes in the local hydration of the phosphodiester group. This is consistent with the fact that there is an inverse correlation between the hydrogen-bond-donating ability of the solvents and the magnitude of the shifts they induce. Other possible interpretations, such as solvent-induced ion pairing and solvent-induced conformational changes, appear to be eliminated. Fourier-transform ir study of the cyclic nucletides reveals that there are also large solvent-induced shifts in the frequency of the antisymmetric OPO stretching frequency, and a comparison of the two types of measurements indicates that there is a linear correlation between shifts observed in the ir and in the ³¹P-nmr spectra. With UpU, the solvent-induced ³¹P-nmr shifts are ～3 times smaller than those observed with the cyclic phosphates and the solvent-induced shift of the OPO band is reduced (factor of ～1.7) as compared with the cyclic phosphates. With the single-stranded polynuclotides, poly(C) and poly(U), the solvent-induced shifts in both the nmr and ir are quite small (～0.1 ppm and ～1 cm^?1). The very small solvent effects observed with poly(U) and poly(C) are attributed to a combination of steric effects and a polyelectrolyte effect which maintains a high density of counterions with waters of hydration in the vicinity of the charged backbone and makes the phosphates much less susceptible to solvent-induced changes in hydration.     

The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs‐Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔC_p) of folding. If increased or enhanced non‐covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow–Klentaq homologous pair, the folding enthalpy (ΔH_fold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (ΔG_fold) originates from a significantly reduced entropic penalty of folding (ΔS_fold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic–thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar “reduced ΔC_p mechanism” for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced‐entropic‐penalty model. Proteins 2014; 82:785–793. © 2013 Wiley Periodicals, Inc.     

Ulva: An emerging green seaweed model for systems biology

Green seaweeds exhibit a wide range of morphologies and occupy various ecological niches, spanning from freshwater to marine and terrestrial habitats. These organisms, which predominantly belong to the class Ulvophyceae, showcase a remarkable instance of parallel evolution toward complex multicellularity and macroscopic thalli in the Viridiplantae lineage. Within the green seaweeds, several Ulva species (“sea lettuce”) are model organisms for studying carbon assimilation, interactions with bacteria, life cycle progression, and morphogenesis. Ulva species are also notorious for their fast growth and capacity to dominate nutrient-rich, anthropogenically disturbed coastal ecosystems during “green tide” blooms. From an economic perspective, Ulva has garnered increasing attention as a promising feedstock for the production of food, feed, and biobased products, also as a means of removing excess nutrients from the environment. We propose that Ulva is poised to further develop as a model in green seaweed research. In this perspective, we focus explicitly on Ulva mutabilis/compressa as a model species and highlight the molecular data and tools that are currently available or in development. We discuss several areas that will benefit from future research or where exciting new developments have been reported in other Ulva species.     

Dissecting the non-specific and specific components of the initial folding reaction of barstar by multi-site FRET measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38-55 and 51-69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein (U) to unfolded protein in refolding conditions (U(C)). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate (I(E)). FRET efficiency and distance change differently for the different donor-acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U(C) and I(E) does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U(C) and to I(E) are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I(E), possesses specific structure.     

Chronic inflammation in mice exposed to the long-term un-entrainable light–dark cycles

Shift work is indispensable in our modern society; therefore, measures for maintaining the health of shift workers are urgently required. In this prospective study observing the effects of different types of scheduled light–dark shift conditions in wild-type mice, we reared mice for 630 days under two chronic jet lag conditions with distinct programs of light and darkness: an 8-h phase delay every 7 days (delay, n = 14) or an 8-h phase advance every 4 days (advance, n = 34). “Delay” represents a mild condition in which mice are entrained, whereas “Advance” represents a condition of circadian rhythm disorder in which mice are not entrained. Our results showed that the 26.5% (9/34) mice reared under the “Advance” shift condition died or were sacrificed by humane endpoint throughout the experiment, whereas the first death under “Delay” condition occurred on day 611. The mortality rate of the mice reared under “Advance” condition was higher than the mice under “Delay” condition [HR (95% CI) 4.26 (0.54–33.6)]. In “Advance” mice of shorter lifespan, which showed splenomegaly and increased myeloid cells in bone marrow, which indicates that chronic inflammation existed in those mice. Although this study is still preliminary, these findings suggest that prolonged severe circadian rhythm disorder may result in the induction of chronic inflammation and tend to result in a shorter lifespan. Further studies are needed to understand why and how circadian rhythm disorder increases the risk of various dysfunction of physiology and diseases.

     

Uncoupled binding and folding of immune signaling-related intrinsically disordered proteins

The classical protein structure-function paradigm has been challenged by the emergence of intrinsically disordered proteins (IDPs), the proteins that do not adopt well-defined three-dimensional structures under physiological conditions. This development was accompanied by the introduction of a “coupled binding and folding” paradigm that suggests folding of IDPs upon binding to their partners. However, our recent studies challenge this general view by revealing a novel, previously unrecognized phenomenon – uncoupled binding and folding. This biologically important mechanism is characteristic of members of a new family of IDPs involved in immune signaling and underlies their unusual properties including: (1) specific homodimerization, (2) the lack of folding upon binding to a well-folded protein, another IDP molecule, or to lipid bilayer membranes, and (3) the “scissors-cut paradox”. The third phenomenon occurs in diverse IDP interactions and suggests that properties of IDP fragments are not necessarily additive in the context of the entire protein. The “no disorder-to-order transition” type of binding is distinct from known IDP interactions and is characterized by an unprecedented observation of the lack of chemical shift and peak intensity changes in multidimensional NMR spectra, a fingerprint of proteins, upon complex formation. Here, I focus on those interactions of IDPs with diverse biological partners where the binding phase driven by electrostatic interactions is not be necessarily followed by the hydrophobic folding phase. I also review new multidisciplinary knowledge about immune signaling-related IDPs and show how it expands our understanding of cell function with multiple applications in biology and medicine.     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist’s view of PrP functions might be clearer at a greater phylogenetic distance.     

Discrimination of Ipomoea aquatica cultivars and bioactivity correlations using NMR-based metabolomics approach

Ipomoea aquatica Forsk is a green leafy vegetable that is a rich source of minerals, proteins, vitamins, amino acids, and secondary metabolites. Different types of I. aquatica cultivars are grown for consumption but little is known about the metabolites variation. Proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with multivariate data analysis was applied for metabolic profiling of three I. aquatica cultivars including “broad leaf (K-25)”, “bamboo leaf (K-88)”, and “special pointed leaf (K-11)”. The orthogonal partial least squares discriminant analysis (OPLS-DA) indicated a clear separation among cultivars. The relative levels of various compounds, such as amino acids, organic acids, sugars, and phenolic compounds were specific to each cultivar. The K-11 cultivar was different from the other cultivars due to a high phenolic content. The content of sugars and some amino acids was higher in K-88 and K-25 possessed a higher content of organic acids. The in vitro study revealed that the I. aquatica cultivars exhibited potent antioxidant and α-glucosidase activities. The results of this study indicate that the K-11 cultivar was the most active due to the abundance of epicatechin, 4,5-dicaffeoylquinic, protocatechuic acid, and rutin.     

Extending the quasi-neutral concept

In caricature, the equilibrium paradigm of community ecology states that plant communities are stable entities consisting of competing species - and that such species coexist because each has its “niche”. This paradigm, in its extreme, has been dead for some time. Nevertheless, it has yet to be replaced by a credible “non” — equilibrium paradigm. The quasi-neutral concept of plant communities, proposed by Kristjan Zobel,Folia Geobot. 36: 3–8, 2001, possesses some of the key ingredients of a nonequilibrium theory of diversity. It recognizes that there are inescapable relationships between diversity at different scales, that similarity can influence the rate of competitive exclusion, that successional change is typically associated with changes in life form, and that rarefaction (i.e. “sampling artifacts”) can have strong effects on fine-scale diversity. However, the current formulation of the quasi-neutral concept is incomplete in that it relies on an unrealistic definition of community, it assumes that random sampling means that species richness at one scale will be linearly related to richness at finer scales, it ignores the possibility of fine-scale processes producing broader-scale patterns, and it avoids the subject of fine-scale environmental heterogeneity. But the most serious limitation of the quasi-neutral concept is that similarity of species alone is not sufficient to allow indefinite coexistence. I present the results of a simple simulation to demonstrate that: (1) identical species will eventually be lost from communities due to stochastic “drift”, (2) slight variations in reproductive rates accelerate this loss, but (3) adding a miniscule “cost of commonness” to the model allows the indefinite coexistence of species. I conclude that the quasi-neutral model cannot work without some kind of trade-off.     

A review of protein engineering for the food industry

In this review I briefly describe the technique of protein engineering and indicate how the present state of knowledge allows proteins to be mutated to increase or decrease stability. I discuss experiments on both model proteins and those of relevance to the food industry and show how hydrophobic forces are a major driving force for folding as well as having a major role in thermostability, I also indicate the large contribution that hydrogen bonding, electrostatic interactions and, in a less well predicted way, disulfide bridges make to thermostability.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Genesis of tramsmissible protein states via deformed templating

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP^Sc and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP^Sc and lack any detectable PrP^Sc particles. Here we discuss a new replication mechanism designated as “deformed templating,” according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.     

The Proteome Folding Problem and Cellular Proteostasis

Stunning advances have been achieved in addressing the protein folding problem, providing deeper understanding of the mechanisms by which proteins navigate energy landscapes to reach their native states and enabling powerful algorithms to connect sequence to structure. However, the realities of the in vivo protein folding problem remain a challenge to reckon with. Here, we discuss the concept of the “proteome folding problem”—the problem of how organisms build and maintain a functional proteome—by admitting that folding energy landscapes are characterized by many misfolded states and that cells must deploy a network of chaperones and degradation enzymes to minimize deleterious impacts of these off-pathway species. The resulting proteostasis network is an inextricable part of in vivo protein folding and must be understood in detail if we are to solve the proteome folding problem. We discuss how the development of computational models for the proteostasis network’s actions and the relationship to the biophysical properties of the proteome has begun to offer new insights and capabilities.