De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

De novo design of protein homodimers containing tunable symmetric protein pockets.

Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.

Structural basis for isoform-specific inhibition of human CTPS1.

Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.

Father- and Mother-Child Reminiscing About Past Pain and Young Children's Cognitive Skills.

Objective Painful experiences are common, distressing, and salient in childhood. Parent-child reminiscing about past painful experiences is an untapped opportunity to process pain-related distress and, similar to reminiscing about other distressing experiences, promotes children's broader development. Previous research has documented the role of parent-child reminiscing about past pain in children's pain-related cognitions (i.e., memories for pain), but no study to date has examined the association between parent-child reminiscing about past painful experiences and children's broader cognitive skills. Design and Methods One hundred and ten typically developing four-year-old children and one of their parents reminisced about a past painful autobiographical event. Children then completed two tasks from the NIH Toolbox Cognitive Battery, the Flanker Inhibitory Control & Attention Test and the Picture Sequence Memory Test, to measure their executive function and episodic memory, respectively. Results Results indicated that the relation between parental reminiscing style and children's executive function was moderated by child sex, such that less frequent parental use of yes-no repetition questions was associated with boys' but not girls', greater performance on the executive function task. Children displayed greater episodic memory performance when their parents reminisced using more explanations. Conclusions The current study demonstrates the key role of parent-child reminiscing about pain in children's broader development and supports the merging of developmental and pediatric psychology fields. Future longitudinal research should examine the directionality of the relation between parent-child reminiscing about past pain and children's developmental outcomes.

The Socialization of Young Children's Empathy for Pain: The Role of Mother- and Father-Child Reminiscing.

OBJECTIVE: Empathy for pain allows one to recognize, understand, and respond to another person's pain in a prosocial manner. Young children develop empathy for pain later than empathy for other negative emotions (e.g., sadness), which may be due to social learning. How parents reminisce with children about past painful events has been linked to children's pain cognitions (e.g., memory) and broader socioemotional development. The present study examined how parent-child reminiscing about pain may be linked to children's empathic behaviors toward another person's pain. METHODS: One hundred and fourteen 4-year-old children (55% girls) and for each, one parent (51% fathers) completed a structured narrative elicitation task wherein they reminisced about a past painful autobiographical event for the child. Children were then observed responding in a lab-based empathy task wherein they witnessed a confederate pretending to hurt themselves. Children's empathic behaviors and parent-child narratives about past painful events were coded using established coding schemes. RESULTS: Findings revealed that parents who used more neutral emotion language (e.g., How did you feel?) when discussing past painful events had children who exhibited more empathic concern in response to another's pain. Similarly, children who used more explanations when reminiscing about past painful events displayed more empathic concern about another's pain. CONCLUSIONS: Findings highlight a key role of parent-child reminiscing about the past pain in the behavioral expression of empathy for pain in young children.

A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity.

The endothelial receptor tyrosine kinase Tie2 plays an important role in vascular formation and maintenance. Mutations in Tie2 lead to vascular malformations, which are painful vascular lesions that cause disfigurement, bleeding, and thrombosis. R849W Tie2 is the most common mutation implicated in an inherited form of vascular malformations and has been shown to be activating, though little is known about the kinetic features of catalysis. Here we undertake a steady-state kinetic analysis of heterologously expressed and purified wild type (WT) and R849W Tie2. While the catalytic efficiencies of the two forms are not significantly different, the observed maximal rate of phosphorylation, kcat,obs, is > 3-fold higher for R849W Tie2 compared to WT. Notably, steady-state catalysis by R849W Tie2 has more striking sigmoidal features compared to WT, suggesting enhanced positive cooperativity. We propose that activating catalytic features are one important consequence of the R849W mutation, though likely other factors such as increased protein binding affinity also contribute to the phenotypes observed in patients.

Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.

Stepwise design of pseudosymmetric protein hetero-oligomers.

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign problems, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 15 new homo-oligomers and recombined them to make 17 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system share a common backbone, and hence are ideal building blocks for generating and functionalizing larger symmetric assemblies.

De novo design of energy transfer proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a "special pair" of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independent of complexities of native photosynthetic proteins, and as a first step towards synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that precisely position chlorophyll dimers. X-ray crystallography shows that one designed protein binds two chlorophylls in a binding orientation matching native special pairs, while a second positions them in a previously unseen geometry. Spectroscopy reveals excitonic coupling, and fluorescence lifetime imaging demonstrates energy transfer. We designed special pair proteins to assemble into 24-chlorophyll octahedral nanocages; the design model and cryo-EM structure are nearly identical. The design accuracy and energy transfer function of these special pair proteins suggest that de novo design of artificial photosynthetic systems is within reach of current computational methods.

Let's (Not) Talk About Pain: Mothers' and Fathers' Beliefs Regarding Reminiscing About Past Pain.

Children's memories for past pain set the stage for their future pain experiences. Parent-child reminiscing about pain plays a key role in shaping children's pain memories. Parental beliefs about the functions of reminiscing are associated with parental reminiscing behaviors. To date, no studies have investigated parental beliefs regarding the functions of reminiscing about past pain or the associations between parental beliefs and reminiscing about past pain. This study aimed to fill these gaps. One-hundred and seven parents (52% fathers) of young children were asked about their beliefs regarding reminiscing about past pain. Interview data were first analyzed using inductive reflexive thematic analysis. A coding scheme was created based on the generated themes to quantitatively characterize parental beliefs. Parents also reminisced with their children about unique past events involving pain. Parent-child reminiscing narratives were coded to capture parent reminiscing behaviors. Inductive reflexive thematic analysis generated three major themes representing parental beliefs regarding reminiscing about past pain: "reminiscing to process past pain," "reminiscing as a learning tool," and "avoiding reminiscing about past pain." Parents who endorsed avoiding reminiscing used fewer optimal reminiscing elements (i.e., open-ended questions) when reminiscing about past painful experiences with children. Parents who endorsed reminiscing to process past pain used more emotion-laden language when reminiscing about past pain. Mothers and fathers of boys and girls endorsed the reminiscing functions to a similar degree. Parents of older, vs. younger, children endorsed reminiscing to process past pain to a greater degree. Developmental considerations and clinical implications of the findings are discussed.

Portrayals of Pain in Children's Popular Media: Mothers' and Fathers' Beliefs and Attitudes.

Evidence suggests that children's popular media may model maladaptive and distorted experiences of pain to young children. In a recent study, pain depicted in popular media targeting 4-6-year-olds was frequently and unrealistically portrayed, evoked little response or empathy from observing characters, and perpetuated unhelpful gender stereotypes. Parents play a critical role in both children's pain experiences and children's media consumption. Yet, no study to date has examined parents' beliefs and attitudes regarding how pain is portrayed in media for young children. The present study aimed to fill this gap by examining how parents perceive and appraise painful instances depicted in children's popular media. Sixty parents (48% fathers) of children aged 4 to 6 years completed a semi-structured interview to assess their general beliefs and attitudes toward how pain is portrayed in children's media. Inductive reflexive thematic analysis was conducted to identify and analyze key patterns in the data. Qualitative analyses generated two major themes representing parental beliefs regarding pain that is portrayed in children's media: "entertaining pain" and "valuable lessons". Findings reveal that parents believe that pain portrayed in popular media serves either a function of entertaining and amusing children or can provide valuable lessons about appropriate emotional responses and empathic reactions. Further, pain portrayals could also instill valuable lessons and provide children with a point of reference and language for their own painful experiences. Parents serve as a primary socialization agent for young children; thus, it is important that parents remain aware of underlying messages about how pain is portrayed in children's popular media so that they can optimally discuss these portrayals, promote their children's pain education and understanding and positively impact future pain experiences.

Reframe the Pain: A Randomized Controlled Trial of a Parent-Led Memory-Reframing Intervention.

Negatively-biased pain memories (ie, recalling more pain as compared to earlier reports) are a robust predictor of future pain experiences. This randomized controlled trial examined the efficacy of a memory-reframing intervention to reframe children's pain memories. Sixty-five children (54% girls, Mage=5.35 years) underwent a tonsillectomy and reported their levels of post-surgical pain intensity and pain-related fear. 2 weeks post-surgery, children and 1 of their parents were randomized to the memory-reframing intervention or control group. Following control/intervention instructions, parents and children reminisced about the past surgery as they normally would (control) or using the memory-reframing strategies (intervention). Children recalled their post-surgical pain intensity and pain-related fear one week later. Parents reported the intervention's acceptability. Recruitment statistics were used to assess feasibility. Controlling for initial pain intensity ratings and using the Faces Pain Scale Revised, children in the intervention group reported more accurate/positively-biased memories for day 1 post-surgery pain intensity (M = 2.60/10; 95% CI, 1.62 to 3.68), compared to children in the control group (M = 4.11/10; 95% CI, 3.12 to 5.03), ηp2 = .07, p = .040. The intervention was acceptable and feasible. Optimal parent-child reminiscing about a past pain experience resulted in children remembering their pain more accurately/positively. Clinicaltrials.gov:NCT03538730. PERSPECTIVE: This article presents results of the first randomized controlled trial examining the efficacy of parent-led memory-reframing intervention to change children's memories for pain. Children of parents who were taught and engaged in optimal reminiscing about a past surgery experience remembered their pain intensity more accurately/positively.

The sociocultural context of pediatric pain: an examination of the portrayal of pain in children's popular media.

ABSTRACT: Pain (eg, needle injections, injuries, and chronic pain) is highly prevalent in childhood and occurs in social contexts. Nevertheless, broader sociocultural influences on pediatric pain, such as popular media, have not been empirically examined. This study examined how pain is portrayed and gendered in children's popular media. A cross-section of children's media targeted towards 4- to 6-year-old children was selected based on popularity, including 10 movies and the first season of 6 television shows. Pain instances were extracted and coded using 2 established observational coding systems assessing sufferer pain characteristics and observer responses (eg, empathic responses). Findings identified 454 instances of pain across the selected media. Violent pain (ie, intentionally inflicted) and injuries were most commonly represented, whereas everyday, chronic-type, and procedural pains were infrequently portrayed. Pain instances were more commonly experienced by boy characters, who also expressed greater distress; yet, observers were more responsive (eg, expressed greater concern) towards girl characters' pain. Overall, observer responses to pain were infrequent, with observers witnessing but not responding to nearly half of pain instances. Observers who did respond expressed an overall lack of empathy towards sufferers. These findings reveal a very narrow depiction of pain presented in children's popular media, with an overall underrepresentation of pain, numerous maladaptive portrayals of pain, and gender differences in both sufferer and observer responses. This study underscores the need for further research to inform how children's popular media is perceived by parents and children and how media may be transformed and harnessed for effective pain education in childhood.

Design of functionalised circular tandem repeat proteins with longer repeat topologies and enhanced subunit contact surfaces.

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.