Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration–approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family.Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, chronic fibrotic and inflammatory diseases, and tumor invasion and metastasis (1–6). These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7–16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.PAI-1 is the most potent physiologic inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively) (17). Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19). However, before the protease can dissociate from the serpin, a dramatic conformational change occurs [termed the stressed to relaxed (S to R) transition], in which the cleaved RCL inserts into the central β-sheet A, translocating the covalently-bound protease 70 Å to the base of β-sheet A (20). This conformational change results in distortion of the protease active site (21, 22) and thereby prevents the deacylation reaction, inactivating the protease. Should this conformational change be interrupted, the protease can complete its cleavage of the serpin RCL, remaining active and leaving the serpin in a cleaved, inactive, loop-inserted state (23).PAI-1 is unique among serpins in that it readily autoinactivates into a so-called latent form, where the PAI-1 RCL inserts into the central A-sheet without cleavage (24). In the latent form, the scissile bond is inaccessible to target proteases and PAI-1’s affinity for vitronectin is greatly reduced (25). Under physiological conditions, the latency transition is irreversible; however, denaturant-induced refolding can reconstitute PAI-1 in the native, active form (26). Several latency-inducing agents have been described, including amphipathic nonionic detergents (27, 28) and monoclonal antibodies or peptides (29–31). These latter agents are thought to stabilize a prelatent intermediate and thereby shift the structural equilibrium toward the latent conformation. None of these agents have been further developed as therapeutics.Such a complex antiproteolytic mechanism, requiring global conformational changes in the serpin structure, as well as PAI-1’s conformational instability, suggests that it should be possible to inhibit the serpin mechanism in multiple ways. Indeed, it is anticipated that minor disruptions in the conformational change may be sufficient to prevent trapping of the protease. This complexity is unlike an enzyme with a single active site, or a receptor with a single ligand binding site, and it may be that the structurally dynamic nature of serpins predisposes anti-PAI-1 high-throughput screens to identifying relatively weak affinity or nonspecific small molecules. Furthermore, the transient nature of the native conformation in the absence of the cofactor vitronectin complicates in vitro screening and therapeutic development (32).We report here the biochemical and structural characterization of a unique high-affinity synthetic PAI-1 inactivator, CDE-096, that is based on our previously described class of polyphenolic PAI-1 inhibitors (33). These inactivators are the first known serpin antagonists with a mechanism of action that involves reversible modulation of the RCL conformation to block Michaelis complex formation. Significantly, CDE-096 is active against both free PAI-1 and vitronectin-bound PAI-1. CDE-096 binds to PAI-1 with nanomolar affinity and induces conformational changes that prevent binding to both proteases and vitronectin. This allosteric mechanism is characterized by reciprocal communication between the high-affinity compound-binding and vitronectin-binding sites. Remarkably, our structural studies suggest a binding site for CDE-096 at the interface between the B and C β-sheets (termed the sB/sC pocket). Although the overall fold of serpins are generally conserved, the sB/sC pocket exhibits considerable structural and sequence variability across the serpin superfamily, thus providing a means of target specificity. Taken together, these data establish a unique mechanistic class of PAI-1 antagonists and provide a distinct binding site for the rational design of future anti-serpin agents.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Solid-phase peptide synthesis and solid-phase fragment coupling mediated by isonitriles

The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this chemistry to reiterative solid-phase peptide synthesis as well as solid-phase fragment coupling are described.Amide bond formations are arguably among the most important constructions in organic chemistry (1, 2). The centrality of the amide linkage, as found in polypeptides and proteins, in the maintenance of life hardly needs restatement. Numerous strategies, resulting in a vast array of protocols to synthesize biologically active polypeptides and proteins, have been demonstrated (3, 4). Central to reiterative polypeptide bond formations was the discovery and remarkable development of solid-phase peptide synthesis (SPPS) (5, 6). The extraordinary impact of SPPS in fostering enhanced access to homogeneous polypeptides is clear to everyone in the field.As we have described elsewhere, by classical, mechanistic reasoning, we were led to conjecture about some hitherto-unexplored possibilities relevant to the chemistry of isonitriles (7–14). It was anticipated that isonitriles might be able to mediate the acylation of amines, thus giving rise to amides (15). Early experiments focused on free carboxylic acids as the acylating agents. As our studies progressed, it was found that the combination of thioacids, amines, and isonitriles leads to the efficient formation of amide bonds under stoichiometric or near-stoichiometric conditions (7–13, 16, 17). Although there remain unresolved issues of detail and nuance, the governing mechanism for amide formation under these conditions involves reaction of the thioacid, 1, with an isonitrile, 2, to generate a thio-formimidate carboxylate mixed anhydride (thio-FCMA), 3, which is intercepted by the “acyl-accepting” amine to generate amide, 5, and thioformamide, 6 (Fig. 1). The efficiency of the amidation was further improved through the use of hydroxybenzotriazole (HOBt) (18), which could well give rise to HOBt ester 7, although this pathway has not been mechanistically proven.Open in a separate windowFig. 1.Isonitrile-mediated amidation; structure of OT.The potentialities of the isonitrile-mediated amidation method were foreshadowed via its application to the synthesis of cyclosporine (19). The power of the method was particularly well demonstrated in the context of our recent total synthesis of oxytocin (OT) (20), wherein isonitrile mediation was used in each of the peptide bond constructions, leading to the synthesis of the hormone in high yield and excellent purity. This nonapeptide is involved in a range of biological functions including parturition and lactation (21, 22). Signaling of OT to its receptor (OTR) is apparently an important factor in quality maintenance of various CNS functions (23). The ability to synthesize such modestly sized, but bio-impactful peptides in both native (wild-type) form, and as strategically modified variants, is one of the current missions of our laboratory, with the objective of possible applications to the very serious problem of autism (24–26).     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.     

Microscopic identification of the order parameter governing liquid–liquid transition in a molecular liquid

A liquid–liquid transition (LLT) in a single-component substance is an unconventional phase transition from one liquid to another. LLT has recently attracted considerable attention because of its fundamental importance in our understanding of the liquid state. To access the order parameter governing LLT from a microscopic viewpoint, here we follow the structural evolution during the LLT of an organic molecular liquid, triphenyl phosphite (TPP), by time-resolved small- and wide-angle X-ray scattering measurements. We find that locally favored clusters, whose characteristic size is a few nanometers, are spontaneously formed and their number density monotonically increases during LLT. This strongly suggests that the order parameter of LLT is the number density of locally favored structures and of nonconserved nature. We also show that the locally favored structures are distinct from the crystal structure and these two types of orderings compete with each other. Thus, our study not only experimentally identifies the structural order parameter governing LLT, but also may settle a long-standing debate on the nature of the transition in TPP, i.e., whether the transition is LLT or merely microcrystal formation.Liquid-liquid transition (LLT) is an intriguing phenomenon in which a liquid transforms into another one via a first-order transition. This means that there can be more than two liquid states for a single-component substance. Despite its counterintuitive nature, there have recently been many pieces of experimental and numerical evidence for the existence of LLT, for various liquids such as water (1–5), aqueous solutions (6–8), triphenyl phosphite (9–12), l-butanol (13), phosphorus (14), silicon (15, 16), germanium (17), and Y₂O₃–Al₂O₃ (18, 19). This suggests that the LLT may be rather universally observed for various types of liquids. However, none of the LLTs reported so far is free from criticisms (20, 21), mainly because these LLTs take place under experimentally difficult conditions [e.g., at high temperature and pressure (14, 15, 17–19)] or in a supercooled state below the melting point (1–3, 5–7, 9, 10), where the transition is inevitably contaminated by microcrystal formation. The latter is not limited to experiments but arises in numerical simulations, often causing many controversies [LLT (22–25) vs. crystallization (26–28)]. For ST2 water, however, this issue has recently been settled by an extensive simulation study by Palmer et al. (4).One of the hottest and long-standing debates is on the nature of the transition found in a molecular liquid, triphenyl phosphite (TPP), by Kivelson and his coworkers (29). The transition is very easy to access experimentally, because it takes place at ambient pressure and at a temperature range between 230 and 210 K and the transformation speed is slow enough to follow the kinetics. Since the finding of this transition (29, 30), many researchers thus have been interested in this intriguing phenomenon and there have been hot discussions on the nature of the transition (20, 21). Some people interpreted this as a liquid-associated phenomenon (9, 10, 31, 32), but others interpret it differently. All of the controversies come from the fact that this transition accompanies microcrystal formation and thus the final state, which is called “glacial phase,” often contains microcrystallites. This led many researchers to explain the transition by non-LLT scenarios, which include a defect-ordered phase scenario predicted by a frustration limited domain theory (29, 30, 33, 34), a microcrystallization scenario (35–38), and a liquid-crystal or plastic-crystal phase scenario (39). Each scenario captures a certain feature of the glacial phase, but fails in explaining all of the experimental results in a consistent manner. Similar situations are often seen in other candidates of LLTs, such as l-butanol [LLT (13) vs. microcrystallization (40–43)], confined water [LLT (5) vs. other phenomena (44–46)], and aqueous solutions [LLT (6, 7) vs. microcrystallization (8, 28, 47, 48)]. For TPP, however, some pieces of experimental evidence supportive of the LLT scenario rather than the microcrystallization scenario have recently been reported (11, 12).We propose a two-order-parameter (TOP) model of a liquid to explain LLT (20, 49). The main point of this model is that it is necessary to consider the spatiotemporal hierarchical nature of a liquid to understand LLT. More specifically, we argue that in addition to density order parameter ρ describing a gas–liquid transition, we need an additional scalar order parameter S, which is the number density of locally favored structures (LFS). In this model, LLT is a consequence of the cooperative ordering of the scalar nonconserved order parameter S, i.e., the cooperative formation of LFS. In other words, LLT is regarded as a gas–liquid-like transition of LFS: one liquid is a gas state of LFS (low-S state), and the other is its liquid state (high-S state). Recently, it was proposed by Anisimov and coworkers (50, 51) that the thermodynamic ordering field conjugate to the order parameter is the conversion equilibrium constant, which further characterizes the nature of LLT. We explained our experimental observation of LLT in TPP in terms of this model (9, 10). We also studied the phase transition dynamics and the physical and chemical properties of the second liquid state (liquid II), which were also explained by the model (20, 21).However, we have not had any direct experimental evidence for the formation of such LFS up to now; thus, an open question is, what is the relevant order parameter governing LLT, although the link of the order parameter to the enthalpy (9, 10), the refractive index (or, density) (9, 10, 29, 30), and the polarity associated with local molecular ordering (12) has been suggested for LLT in TPP. There have been structural studies on LLT by X-ray and neutron scattering measurements, focusing on local liquid structures at an inter- and intramolecular scale (36, 38, 52–54) and mesoscopic structures (34, 55). However, there has been no experimental evidence for the presence of locally favored structures, which characterize the liquid state uniquely, or the order parameter has still not been identified from a microscopic viewpoint.Here we study the structural change of TPP during LLT by time-resolved small- and wide-angle X-ray scattering measurements, which cover a length scale from a single molecule size ( ～  1 nm) to more than tens of nanometers. We show, to our knowledge, the first direct evidence for the presence of LFS and the temporal increase upon the liquid I-to-liquid II transformation. Furthermore, we also find an indication of the formation of microcrystallites during LLT. However, we reveal that LFS and microcrystallites have different sizes and growth kinetics, indicating that although they sometimes appear simultaneously during the process of LLT, LLT itself is driven by the formation of LFS and not by that of microcrystallites. We also discover that LFS are destroyed upon crystallization, clearly indicating not only that these two types of orderings are competing with each other but also that LFS is a structure unique to the liquid state. Our findings provide a comprehensive view on the long-standing controversy on the origin of the glacial phase, which was discovered by Kivelson and his coworkers (29, 30), and show that the fraction of LFS may be the relevant order parameter of LLT. This suggests that a liquid can have a spatiotemporal hierarchical structure at a low temperature, contrary to the common picture of a high-temperature liquid where the structure is random and homogeneous beyond the molecular size.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

Multiaddressable molecular rectangles with reversible host–guest interactions: Modulation of pH-controlled guest release and capture

A series of multiaddressable platinum(II) molecular rectangles with different rigidities and cavity sizes has been synthesized by endcapping the U-shaped diplatinum(II) terpyridine moiety with various bis-alkynyl ligands. The studies of the host–guest association with various square planar platinum(II), palladium(II), and gold(III) complexes and the related low-dimensional gold(I) complexes, most of which are potential anticancer therapeutics, have been performed. Excellent guest confinement and selectivity of the rectangular architecture have been shown. Introduction of pH-responsive functionalities to the ligand backbone generates multifunctional molecular rectangles that exhibit reversible guest release and capture on the addition of acids and bases, indicating their potential in controlled therapeutics delivery on pH modulation. The reversible host–guest interactions are found to be strongly perturbed by metal–metal and π–π interactions and to a certain extent, electrostatic interactions, giving rise to various spectroscopic changes depending on the nature of the guest molecules. Their binding mode and thermodynamic parameters have been determined by 2D NMR and van’t Hoff analysis and supported by computational study.The study of metal–metal interactions has drawn enormous attention since the past two decades because of the intriguing spectroscopic and photophysical properties arising from the close proximity of the metal centers (1, 2). Square planar d⁸ platinum(II) complexes with coordination unsaturation are one of the important classes of metal complexes that have been extensively explored because of their capability to exhibit metal–metal interactions and display rich photophysical properties (3–26). Platinum(II) terpyridine complexes have been found to exhibit rich polymorphism in the solid state (16–20) owing to their square planar coordination geometry, which permits facile access to Pt(II)···Pt(II) interactions as well as π–π interactions between the chromophores. It was not until 2001 that the first successful synthesis of platinum(II) terpyridine alkynyl complexes, which possess enhanced solubility and luminescence compared with the chloro counterpart, was reported (16). Additional efforts have been devoted to the use of the system to respond to external stimuli, such as variation in solvent composition (17, 18), pH (19, 20), temperature (21, 22), addition of ionic (24–26), and polymeric species (27, 28), in which spectral changes induced by strong Pt(II)···Pt(II) and π−π interactions have been displayed.In the past few decades, enormous efforts have been devoted to the construction of molecular architectures by fusing the organic framework to the transition metal centers through self-assembly processes (29–57). There has been continuous interest in the construction of stimuli-responsive metallosupramolecular architectures with diverse sizes, shapes, and symmetries to rationalize the criteria for molecular recognition and impart them on unique areas of applications, such as stereoselective guest encapsulation and molecular transporting devices (45–65). Although such a variety of metal–organic macrocyclic architectures has been reported, those involving the use of noncovalent interactions other than those of hydrogen bonding, donor–acceptor, electrostatic, and hydrophobic–hydrophobic interactions as well as luminescence changes that depend on the nature of the guests, which would be attractive for chemo- and biosensing, have been rare and are rather underexplored. Examples of such systems that can exhibit reversible host–guest association are also limited.Since the discovery of anticancer properties of cisplatin in 1969 (58), the coordination chemistry and the development of related species with enhanced properties and reduced cytotoxicity have received enormous attention. Although the potency and cytotoxicity studies are important, the availability of the drugs and their transport and release to the site of action are equally important. Thus, the design of smart drug delivery systems has been an area of growing interest. The first phosphorescent molecular tweezers making use of the alkynylplatinum(II) terpyridine moiety have been reported by our group to show their host–guest interactions with transition metal complexes (57). However, the opened structures of the tweezers have limited their selectivity and functionality. To accomplish the controlled drug delivery functionalities, the first main strategy is to rigidify the molecular architecture of the host from tweezers to a rectangle, so that the guest molecules would be better accommodated within the cavity, which may lead to a more selective encapsulation of guests within a definite size and steric environment. The possibility of introducing responsive functionalities into the molecular rectangles, which may serve as models for the study of on-demand controlled guest capture and release systems, has also been explored. pH-sensitive pyridine moieties have, therefore, been incorporated into the backbone of the rectangle to modulate the reversible host–guest interaction within the constrained rectangle environment on protonation/deprotonation of the pyridine nitrogen atom to achieve multiaddressable functions that would not have been readily achievable with the molecular tweezers structure. Additionally, the use of various platinum and gold complexes as guest molecules, which have been shown to display anticancer therapeutic behavior (58–65), may lead to the design of a smart multiaddressable molecular rectangle system that could capture and release specific guest molecules under different pH conditions to achieve proof-of-principle on-demand controlled drug delivery. Herein, the design and synthesis of a series of alkynylplatinum(II) terpyridine molecular rectangles (Fig. 1) with different geometries, topologies and electronic properties are reported. Moreover, the encapsulation of various guest molecules is also investigated in detail to provide a proof-of-principle model for the design of artificial drug delivery systems with the modulation of drug release by pH.Open in a separate windowFig. 1.Molecular structures of rectangles 1−4.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.