Design and evolution of artificial enzymes

Protein design is a challenging problem. We do not fully understand the rules of protein folding, and our knowledge of structure-function relationships in these macromolecules is at best incomplete. Nature has solved the problem of protein design through the mechanism of Darwinian evolution. From primitive precursors, recursive cycles of mutation, selection and amplification of molecules with favorable traits have given rise to all of the many thousands of gene products in every one of our cells. An analogous process of natural selection can be profitably exploited in silico and in the laboratory on a human time scale to create, characterize and optimize artificial catalysts for tasks unimagined by Nature. Recent progress in combining computational and evolutionary approaches for enzyme design will be discussed, together with insights into enzyme function gained from studies of the engineered catalysts

Characterization of Cross-Genre Writing Skills in Children with and Without Autism Spectrum Disorders: The Role of Language, Handwriting, and Cognitive Processing

It has been estimated that writing is one of the most significant academic problems for children with Autism Spectrum Disorder (ASD), with as many as 60% of children having a learning disability in writing (Mayes & Calhoun, 2008). The majority of evidence demonstrating this achievement gap, however, comes from research finding global writing deficits, using standardized tests. As a result, a number of questions remain about how the texts constructed by children with ASD specifically align or deviate from typical development. For instance, do these texts differ in terms of vocabulary, grammar, or structure? Are children with ASD better at writing in a particular genre? Additionally, the mechanisms that influence writing development in children with ASD are still unclear. Therefore, in the present study we (1) comprehensively characterized the cross-genre (i.e., personal narrative, expository) writing development of 8- to- 14-year-old children with and without ASD; and (2) examined how language, handwriting ability, and cognitive processing contribute to written expression. Our findings revealed that children with ASD wrote less and made more grammatical errors in their sentences across writing genres than neurotypical (NT) children. When examining overall quality, children with ASD only differed from neurotypical children on their narrative texts. In contrast, writing high quality expository essays was an area of relative strength for children with ASD compared to NT children. Contrary to expectations, children made few significant style distinctions between personal narrative and expository writing. Current analyses also indicated that oral language skills, handwriting ability, theory of mind, and executive functioning each play a role in a variety of written expression skills in children with and without ASD. For example, theory of mind knowledge appeared to be especially important for the quality of writing among children with and without ASD. These results have important implications for educational instruction as well as the development of writing interventions

The enzyme mechanism of a de novo designed and evolved aldolase

The combination of computational enzyme design and laboratory evolution is a successful strategy for the development of biocatalysts with non-natural function, one example being the artificial retroaldolase RA95.1,2 This enzyme utilizes amine catalysis via a reactive lysine residue to cleave the unnatural aldol substrate methodol (Figure 1A). The low initial catalytic activity of the computational design was improved tremendously over many rounds of directed evolution, yielding an efficient biocatalyst for both aldol cleavage as well as synthesis with rate acceleration and stereoselectivity comparable to natural aldolases (Figure 1B).3,4 Key to this success was an ultrahigh-throughput (uHTP) screening technique applied for the late stages of optimization.4 In this work, we analyzed changes in enzyme mechanism along the evolutionary trajectory of RA95 that led to more efficient catalysis. To that end, we determined the rate-limiting step for different enzyme variants by probing individual steps of the aldolase mechanism kinetically. We found a shift towards product release being overall rate-limiting for aldol cleavage catalyzed by highly evolved variants of RA95. Specifically, the conversion between Schiff base and enamine intermediate formed from acetone, a (de-)protonation-dependent process, is the slowest step we probed. Our results indicate that uHTP screening is essential to efficiently evolve a multi-step enzyme mechanism, as it allows the optimization of several mechanistic steps in parallel. By comparing our findings to kinetic and structural studies on natural aldolases, we provide valuable feedback to improve future laboratory evolution approaches as well as the success rate of computational enzyme design. Please click Additional Files below to see the full abstract

Eco-Rehab: An Ecological Guide for the Rehabilitation of Cabins at the Arcola Mills Center

This report was produced as part of a project to help the Arcola Mills Center, which is located along the St Croix River, rehabilitate the cabins on their site in an environmentally responsible fashion. The rehabilitation process was intended to create overnight retreat spaces and included educational features highlighting the historical and environmental aspects of the site and buildings. The intent of the report is to provide guiding principles, a solid body of information on 'green' building, and a general plan for rehabilitating three of the site's buildings--the Mill House, Bungalow, and Houseboat.Prepared in partnership with the Arcola Mills Historic Foundation by the Community Assistantship Program (CAP) administered by the Center for Urban and Regional Affairs, University of Minnesota

Body Image and Life Satisfaction in Amish, Catholic, and Non-Religious Women

Dissatisfaction with one’s appearance is commonplace among Western women. Body image dissatisfaction is believed to be a consequence of societal emphases on appearance reinforced through norms and media. However, some Amish cultural values and norms differ from prevailing Western influences, which may result in a rate of body image dissatisfaction at variance with Western women. The following pilot study explores how religious affiliation and religiosity may relate to body image factors (body dissatisfaction, appearance investment, and body coping strategies) and life satisfaction in Amish (N = 32), Catholic (N = 40), and non-religious (N = 40) women. Results suggest that the Amish have a more positive body image than Catholic women; results were inconclusive when comparing to non-religious women. As this pilot study’s sampling proceduce and size limit our ability to draw definitive conclusions, future research should work toward an expanded, systematic sample. If results from this study are confirmed, research is needed that examines the specific aspects of the Amish culture that may be associated with higher rates of body image satisfaction

A Cognitive-Developmental Approach to Emotion Processing in Children

Although cogmttve development and emotional development are often viewed as separate components in the development of the child, the two are intricately related to each other. The purpose of this chapter is to review and examine how cognitive development is related to emotional development, and how traditional and current theories of cognitive development provide us with a framework for understanding the development of emotion processing in children. Topics explored in this chapter include the role of executive functioning, language development and cognitive concepts of self on the development of emotional competence. Moreover, three theories of cognitive development: Theory of Mind, Weak Central Coherence theory and Systemizing Theory are considered in relation to cognitive and emotional processing in a special population, children with Autism Spectrum Disorders. Finally, this chapter concludes with a discussion of the remaining challenges for theories of cognitive development, especially in terms of exploring the reciprocal relation between cognition and emotion

Examination of Script and Non-Script Based Narrative Retellings in Children with Autism Spectrum Disorders

This study compared the narrative abilities of 19 children with Autism Spectrum Disorder (ASD) and 26 neurotypical children (NT), between 6 and 12 years of age, on two story retelling tasks: a script-based story and a non-script based story. The script- based story contained the structural aspects of a narrative, but also had the internal framework of a script (Hayward et al., 2007). Given the reduced cognitive and linguistic demands of the script-based story, it was expected that the script-based narrative measure would minimize narrative differences between children with and without ASD. Additionally, the relation between narrative production, theory of mind (ToM), and linguistic abilities were examined. Unexpectedly, the narration of both story types was equally difficult for children with ASD for the majority of narrative variables, including syntactic complexity, structure, content, appropriate use of references, and causal connectivity, which resulted in narratives that were less coherent and cohesive than the NT group. Closer examination of the script-based story revealed that children with ASD were including the same number of script details as the NT children, but were less likely to include the non-script details. These findings suggested that the children with ASD had more general narrative impairments, instead of abnormalities in their representation of script knowledge. Among children with ASD, ToM uniquely predicted narrative coherence and cohesion for both story types. Implications for the understanding of common events and the need for narrative interventions are discussed

Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, 2h J NN and 1h J HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic model