TReSR: A PCR-compatible DNA sequence design method for engineering proteins containing tandem repeats.

Protein tandem repeats (TRs) are motifs comprised of near-identical contiguous sequence duplications. They are found in approximately 14% of all proteins and are implicated in diverse biological functions facilitating both structured and disordered protein-protein and protein-DNA interactions. These functionalities make protein TR domains an attractive component for the modular design of protein constructs. However, the repetitive nature of DNA sequences encoding TR motifs complicates their synthesis and mutagenesis by traditional molecular biology workflows commonly employed by protein engineers and synthetic biologists. To address this challenge, we developed a computational protocol to significantly reduce the complementarity of DNA sequences encoding TRs called TReSR (for Tandem Repeat DNA Sequence Redesign). The utility of TReSR was demonstrated by constructing a novel constitutive repressor synthesized by duplicating the LacI DNA binding domain into a single-chain TR construct by assembly PCR. Repressor function was evaluated by expression of a fluorescent reporter delivered on a single plasmid encoding a three-component genetic circuit. The successful application of TReSR to construct a novel TR-containing repressor with a DNA sequence that is amenable to PCR-based construction and manipulation will enable the incorporation of a wide range of TR-containing proteins for protein engineering and synthetic biology applications.

Severe anorectal injury secondary to jet ski accident: An important and increasing mechanism of injury.

Introduction: Traumatic injuries from jet ski-related accidents have increased in incidence over the past few decades. Anorectal injuries are uncommon but typically arise from high-speed jet ski accidents. We present a case of a severe anorectal injury from a fall off the back of an accelerating jet ski. Case report: This case reports on the presentation, operative findings and management of a 22-year-old female with major internal and external anal sphincter disruption sustained via an unusual traumatic mechanism. Operative findings identified a complete internal and external anal sphincter disruption at the 1 and 7 O'clock positions and extra-peritoneal rectal perforation. Washout, suture repair and an end-colostomy were performed. Conclusion: Understanding the potential severity of injury from the insult mechanism is paramount to triaging and managing trauma patients. Although this case describes an inconspicuous mechanism, the resulting trauma is significant and should prompt consideration in future cases. In addition, the article describes an approach to the repair of such injuries and the difficult decision relating to the role and type of defunctioning colostomy to protect any possible missed injuries in a complex traumatic environment, and in the protection of the anorectal repair.

Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget.

Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden - as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.

Rational design of proteins that exchange on functional timescales.

Proteins are intrinsically dynamic molecules that can exchange between multiple conformational states, enabling them to carry out complex molecular processes with extreme precision and efficiency. Attempts to design novel proteins with tailored functions have mostly failed to yield efficiencies matching those found in nature because standard methods do not allow the design of exchange between necessary conformational states on a functionally relevant timescale. Here we developed a broadly applicable computational method to engineer protein dynamics that we term meta-multistate design. We used this methodology to design spontaneous exchange between two novel conformations introduced into the global fold of Streptococcal protein G domain ß1. The designed proteins, named DANCERs, for dynamic and native conformational exchangers, are stably folded and switch between predicted conformational states on the millisecond timescale. The successful introduction of defined dynamics on functional timescales opens the door to new applications requiring a protein to spontaneously access multiple conformational states.

Semantic control deficits impair understanding of thematic relationships more than object identity.

Recent work has suggested a potential link between the neurocognitive mechanisms supporting the retrieval of events and thematic associations (i.e., knowledge about how concepts relate in a meaningful context) and semantic control processes that support the capacity to shape retrieval to suit the circumstances. Thematic associations and events are inherently flexible: the meaning of an item changes depending on the context (for example, lamp goes with reading, bicycle and police). Control processes might stabilise weak yet currently-relevant interpretations during event understanding. In contrast, semantic retrieval for objects (to understand what items are, and the categories they belong to) is potentially constrained by sensory-motor features (e.g., bright light) that change less across contexts. Semantic control and event understanding produce overlapping patterns of activation in healthy participants in left prefrontal and temporoparietal regions, but the potential causal link between these aspects of semantic cognition has not been examined. We predict that event understanding relies on semantic control, due to associations being necessarily context-dependent and variable. We tested this hypothesis in two ways: (i) by examining thematic associations and object identity in patients with semantic aphasia, who have well-documented deficits of semantic control following left frontoparietal stroke and (ii) using the same tasks in healthy controls under dual-task conditions that depleted the capacity for cognitive control. The patients were impaired on both identity and thematic matching tasks, and they showed particular difficulty on non-dominant thematic associations which required greater control over semantic retrieval. Healthy participants showed the same pattern under conditions of divided attention. These findings support the view that semantic control is necessary for organising and constraining the retrieval of thematic associations.

Deconstruction of complex protein signaling switches: a roadmap toward engineering higher-order gene regulators.

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein-ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure-function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI. WIREs Nanomed Nanobiotechnol 2017, 9:e1461. doi: 10.1002/wnan.1461 For further resources related to this article, please visit the WIREs website.

Multistate Computational Protein Design with Backbone Ensembles.

The ability of computational protein design (CPD) to identify protein sequences possessing desired characteristics in vast sequence spaces makes it a highly valuable tool in the protein engineering toolbox. CPD calculations are typically performed using a single-state design (SSD) approach in which amino-acid sequences are optimized on a single protein structure. Although SSD has been successfully applied to the design of numerous protein functions and folds, the approach can lead to the incorrect rejection of desirable sequences because of the combined use of a fixed protein backbone template and a set of rigid rotamers. This fixed backbone approximation can be addressed by using multistate design (MSD) with backbone ensembles. MSD improves the quality of predicted sequences by using ensembles approximating conformational flexibility as input templates instead of a single fixed protein structure. In this chapter, we present a step-by-step guide to the implementation and analysis of MSD calculations with backbone ensembles. Specifically, we describe ensemble generation with the PertMin protocol, execution of MSD calculations for recapitulation of Streptococcal protein G domain ß1 mutant stability, and analysis of computational predictions by sequence binning. Furthermore, we provide a comparison between MSD and SSD calculation results and discuss the benefits of multistate approaches to CPD.

Exploring the role of the posterior middle temporal gyrus in semantic cognition: Integration of anterior temporal lobe with executive processes.

Making sense of the world around us depends upon selectively retrieving information relevant to our current goal or context. However, it is unclear whether selective semantic retrieval relies exclusively on general control mechanisms recruited in demanding non-semantic tasks, or instead on systems specialised for the control of meaning. One hypothesis is that the left posterior middle temporal gyrus (pMTG) is important in the controlled retrieval of semantic (not non-semantic) information; however this view remains controversial since a parallel literature links this site to event and relational semantics. In a functional neuroimaging study, we demonstrated that an area of pMTG implicated in semantic control by a recent meta-analysis was activated in a conjunction of (i) semantic association over size judgements and (ii) action over colour feature matching. Under these circumstances the same region showed functional coupling with the inferior frontal gyrus - another crucial site for semantic control. Structural and functional connectivity analyses demonstrated that this site is at the nexus of networks recruited in automatic semantic processing (the default mode network) and executively demanding tasks (the multiple-demand network). Moreover, in both task and task-free contexts, pMTG exhibited functional properties that were more similar to ventral parts of inferior frontal cortex, implicated in controlled semantic retrieval, than more dorsal inferior frontal sulcus, implicated in domain-general control. Finally, the pMTG region was functionally correlated at rest with other regions implicated in control-demanding semantic tasks, including inferior frontal gyrus and intraparietal sulcus. We suggest that pMTG may play a crucial role within a large-scale network that allows the integration of automatic retrieval in the default mode network with executively-demanding goal-oriented cognition, and that this could support our ability to understand actions and non-dominant semantic associations, allowing semantic retrieval to be 'shaped' to suit a task or context.

Turning a Negative into a Positive: Conversion of a Homodimer into a Heterodimer Using Negative State Repertoires.

In this issue of Structure, Leaver-Fay et al. (2016) engineer bispecific antibodies using multistate computational protein design with negative state repertoires. In combination with additional mutations selected rationally, they produced antibodies that assembled as heterodimers with up to 93% purity.

Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif.

Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.

Diastereoseletive Transannular Oxa-Conjugate Addition Generates the 2,6-cis-Disubstituted Tetrahydropyran of Neopeltolide.

Transannular 2,6-disubstituted pyrans, like the one found in the cytotoxic marine natural product neopeltolide, are a key functional group in many polyketides. While oxa-conjugate additions have been shown to provide direct and rapid access to tetrahydropyrans in acyclic neopeltolide intermediates, a transannular strategy for construction of this ring system in a macrocyclic core has not been investigated. In this study, we demonstrate that a transannular oxa-conjugate addition strategy is a viable approach to the construction of the bicyclic core of neopeltolide. We show that transannular addition occurs readily with an α,ß-unsaturated ketone as the Michael acceptor and does not occur when an α,ß-unsaturated ester is the Michael acceptor. Our data indicates that oxa-conjugate addition is reversible and that the stereochemical outcome can be under thermodynamic control. Using computational chemistry, we show that the lowest energy diastereomer is the desired cis-pyran found in neopeltolide, and we experimentally demonstrate that the trans and cis diastereomers are interconvertible under reaction conditions with the cis-pyran product predominating. This oxa-conjugate addition strategy should provide a viable route to accessing the fully elaborated macrocyclic core of neopeltolide.

Automatic and Controlled Semantic Retrieval: TMS Reveals Distinct Contributions of Posterior Middle Temporal Gyrus and Angular Gyrus.

Semantic retrieval involves both (1) automatic spreading activation between highly related concepts and (2) executive control processes that tailor this activation to suit the current context or goals. Two structures in left temporoparietal cortex, angular gyrus (AG) and posterior middle temporal gyrus (pMTG), are thought to be crucial to semantic retrieval and are often recruited together during semantic tasks; however, they show strikingly different patterns of functional connectivity at rest (coupling with the "default mode network" and "frontoparietal control system," respectively). Here, transcranial magnetic stimulation (TMS) was used to establish a causal yet dissociable role for these sites in semantic cognition in human volunteers. TMS to AG disrupted thematic judgments particularly when the link between probe and target was strong (e.g., a picture of an Alsatian with a bone), and impaired the identification of objects at a specific but not a superordinate level (for the verbal label "Alsatian" not "animal"). In contrast, TMS to pMTG disrupted thematic judgments for weak but not strong associations (e.g., a picture of an Alsatian with razor wire), and impaired identity matching for both superordinate and specific-level labels. Thus, stimulation to AG interfered with the automatic retrieval of specific concepts from the semantic store while stimulation of pMTG impaired semantic cognition when there was a requirement to flexibly shape conceptual activation in line with the task requirements. These results demonstrate that AG and pMTG make a dissociable contribution to automatic and controlled aspects of semantic retrieval. SIGNIFICANCE STATEMENT: We demonstrate a novel functional dissociation between the angular gyrus (AG) and posterior middle temporal gyrus (pMTG) in conceptual processing. These sites are often coactivated during neuroimaging studies using semantic tasks, but their individual contributions are unclear. Using transcranial magnetic stimulation and tasks designed to assess different aspects of semantics (item identity and thematic matching), we tested two alternative theoretical accounts. Neither site showed the pattern expected for a "thematic hub" (i.e., a site storing associations between concepts) since stimulation disrupted both tasks. Instead, the data indicated that pMTG contributes to the controlled retrieval of conceptual knowledge, while AG is critical for the efficient automatic retrieval of specific semantic information.

Prediction of Stable Globular Proteins Using Negative Design with Non-native Backbone Ensembles.

Accurate predictions of protein stability have great potential to accelerate progress in computational protein design, yet the correlation of predicted and experimentally determined stabilities remains a significant challenge. To address this problem, we have developed a computational framework based on negative multistate design in which sequence energy is evaluated in the context of both native and non-native backbone ensembles. This framework was validated experimentally with the design of ten variants of streptococcal protein G domain ß1 that retained the wild-type fold, and showed a very strong correlation between predicted and experimental stabilities (R(2) = 0.86). When applied to four different proteins spanning a range of fold types, similarly strong correlations were also obtained. Overall, the enhanced prediction accuracies afforded by this method pave the way for new strategies to facilitate the generation of proteins with novel functions by computational protein design.

Conceptual control across modalities: graded specialisation for pictures and words in inferior frontal and posterior temporal cortex.

Controlled semantic retrieval to words elicits co-activation of inferior frontal (IFG) and left posterior temporal cortex (pMTG), but research has not yet established (i) the distinct contributions of these regions or (ii) whether the same processes are recruited for non-verbal stimuli. Words have relatively flexible meanings - as a consequence, identifying the context that links two specific words is relatively demanding. In contrast, pictures are richer stimuli and their precise meaning is better specified by their visible features - however, not all of these features will be relevant to uncovering a given association, tapping selection/inhibition processes. To explore potential differences across modalities, we took a commonly-used manipulation of controlled retrieval demands, namely the identification of weak vs. strong associations, and compared word and picture versions. There were 4 key findings: (1) Regions of interest (ROIs) in posterior IFG (BA44) showed graded effects of modality (e.g., words>pictures in left BA44; pictures>words in right BA44). (2) An equivalent response was observed in left mid-IFG (BA45) across modalities, consistent with the multimodal semantic control deficits that typically follow LIFG lesions. (3) The anterior IFG (BA47) ROI showed a stronger response to verbal than pictorial associations, potentially reflecting a role for this region in establishing a meaningful context that can be used to direct semantic retrieval. (4) The left pMTG ROI also responded to difficulty across modalities yet showed a stronger response overall to verbal stimuli, helping to reconcile two distinct literatures that have implicated this site in semantic control and lexical-semantic access respectively. We propose that left anterior IFG and pMTG work together to maintain a meaningful context that shapes ongoing semantic processing, and that this process is more strongly taxed by word than picture associations.

Shared neural processes support semantic control and action understanding.

Executive-semantic control and action understanding appear to recruit overlapping brain regions but existing evidence from neuroimaging meta-analyses and neuropsychology lacks spatial precision; we therefore manipulated difficulty and feature type (visual vs. action) in a single fMRI study. Harder judgements recruited an executive-semantic network encompassing medial and inferior frontal regions (including LIFG) and posterior temporal cortex (including pMTG). These regions partially overlapped with brain areas involved in action but not visual judgements. In LIFG, the peak responses to action and difficulty were spatially identical across participants, while these responses were overlapping yet spatially distinct in posterior temporal cortex. We propose that the co-activation of LIFG and pMTG allows the flexible retrieval of semantic information, appropriate to the current context; this might be necessary both for semantic control and understanding actions. Feature selection in difficult trials also recruited ventral occipital-temporal areas, not implicated in action understanding.

Optimization of rotamers prior to template minimization improves stability predictions made by computational protein design.

Computational protein design (CPD) predictions are highly dependent on the structure of the input template used. However, it is unclear how small differences in template geometry translate to large differences in stability prediction accuracy. Herein, we explored how structural changes to the input template affect the outcome of stability predictions by CPD. To do this, we prepared alternate templates by Rotamer Optimization followed by energy Minimization (ROM) and used them to recapitulate the stability of 84 protein G domain ß1 mutant sequences. In the ROM process, side-chain rotamers for wild-type (WT) or mutant sequences are optimized on crystal or nuclear magnetic resonance (NMR) structures prior to template minimization, resulting in alternate structures termed ROM templates. We show that use of ROM templates prepared from sequences known to be stable results predominantly in improved prediction accuracy compared to using the minimized crystal or NMR structures. Conversely, ROM templates prepared from sequences that are less stable than the WT reduce prediction accuracy by increasing the number of false positives. These observed changes in prediction outcomes are attributed to differences in side-chain contacts made by rotamers in ROM templates. Finally, we show that ROM templates prepared from sequences that are unfolded or that adopt a nonnative fold result in the selective enrichment of sequences that are also unfolded or that adopt a nonnative fold, respectively. Our results demonstrate the existence of a rotamer bias caused by the input template that can be harnessed to skew predictions toward sequences displaying desired characteristics.

Discovery of substrates for a SET domain lysine methyltransferase predicted by multistate computational protein design.

Characterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]-1-K(∗)-[AFYMSHRK]+1-[LYK]+2 around the target lysine K(∗). Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes.

Improving the accuracy of protein stability predictions with multistate design using a variety of backbone ensembles.

Multistate computational protein design (MSD) with backbone ensembles approximating conformational flexibility can predict higher quality sequences than single-state design with a single fixed backbone. However, it is currently unclear what characteristics of backbone ensembles are required for the accurate prediction of protein sequence stability. In this study, we aimed to improve the accuracy of protein stability predictions made with MSD by using a variety of backbone ensembles to recapitulate the experimentally measured stability of 85 Streptococcal protein G domain ß1 sequences. Ensembles tested here include an NMR ensemble as well as those generated by molecular dynamics (MD) simulations, by Backrub motions, and by PertMin, a new method that we developed involving the perturbation of atomic coordinates followed by energy minimization. MSD with the PertMin ensembles resulted in the most accurate predictions by providing the highest number of stable sequences in the top 25, and by correctly binning sequences as stable or unstable with the highest success rate (≈90%) and the lowest number of false positives. The performance of PertMin ensembles is due to the fact that their members closely resemble the input crystal structure and have low potential energy. Conversely, the NMR ensemble as well as those generated by MD simulations at 500 or 1000 K reduced prediction accuracy due to their low structural similarity to the crystal structure. The ensembles tested herein thus represent on- or off-target models of the native protein fold and could be used in future studies to design for desired properties other than stability.