Understanding Stability of Protein-Protein Complexes

For all living organisms, macromolecular interactions facilitate most of their natural functions. Alterations to macromolecular structures through mutations, can affect the stability of their interactions, which may lead to unfavourable phenotypes and disease. Presented here, are a number of computational methods aimed at uncovering the principles behind complex stability - as described by binding affinity and dissociation rate constants. Several factors are known to govern the stability of protein-protein interactions, however, no one factor dominates, and it is the synergistic effect of a number of contributions, which amount to the affinity, and stability of a complex. The characterization of complex stability can thus be presented as a two-fold problem; modelling the individual factors and modelling the synergistic effect of the combination of such individual factors. Using machine learning as a central framework, empirical functions are designed for estimating affinity, dissociation rates and the effects of mutations on these properties. The performance of all models is in turn benchmarked on experimental data available from the literature and carefully curated datasets. Firstly, a wild-type binding free energy prediction model is designed, composed of a diverse set of stability descriptors, which account for flexibility and conformational changes undergone by the complex in question. Similarly, models for estimating the effects of mutations on binding affinity are also designed and benchmarked in a community-wide blind trial. Emphasis here is on the detection of a small subset of mutations that are able to enhance the stability of two de novo protein drugs targeting the flu virus hemagglutinin. Probing further the determinants of stability, a set of descriptors that link hotspot residues with the off-rate of a complex are designed, and applied to models predicting changes in off-rate upon mutation. Finally, the relationship between the distribution of hotspots at protein interfaces, and the rate of dissociation of such interfaces, is investigated

Nitrate metabolism in the Epsilonproteobacteria: Campylobacter jejuni and Sulfurospirillum barnesii

In this study the nitrate metabolism of Campylobacter jejuni and Sulfurospirillum barnesii will be examined, specifically the periplasmic nitrate reductase (Nap) enzyme which is transforms of nitrate to nitrite. The catalytic subunit, NapA, is a molybdenum dependent enzyme. Isolation of molybdenum containing enzymes is not straightforward as co-factor can be lost during protein purification procedures. This study used two protein purification methods to isolate NapA. First, NapA was isolated directly from S. barnesii using protein fractionation and anion exchange chromatography. Second, molecular cloning was used to express the recombinant affinity-tagged S.barnesii and C. jejuni NapA proteins from E. coli. Immobilized metal affinity chromatography was used to isolate the recombinant proteins. NapD was co-expressed with NapA to aid in post-translational modifications. The reduced methyl viologen assay was used to study the kinetics of nitrate reduction. Comparison of the native and recombinant NapA kinetic properties suggests that the recombinant enzyme have attenuated activity. The theoretical structure of C. jejuni NapA was calculated using homology modeling techniques. Comparison of the C. jejuni NapA with structures of NapA from other organisms indicates that C. jejuni NapA has large sequence inserts on the outside of the protein. Furthermore, the napA operon of C. jejuni and S. barnesii display distinct gene content and organization

Cellular and Developmental Insights into the Early Evolution of Muscle

Whereas a great deal has been learned about the molecular underpinnings of morphological evolution in animals, much less is known about the origin of novel cell and tissue types. During the time in which the earliest animal lineages were diversifying, fundamental cell and tissue types, such as muscles, arose. Sponges are one of two animal lineages that lack muscles, yet they undergo coordinated full body contractions. Whereas the signaling processes have been studied, the physical mechanisms of contraction are completely uncharacterized. The main purpose of this work is to understand the primary contractile tissue of the sponge Ephydatia muelleri, from a structural, functional, and developmental standpoint. As there is no single unique feature that is shared by all muscle types in animals, a full picture of the contractile tissue of E. muelleri is needed to assess its homology with these tissues. Here the focus is on an endothelial-like tissue containing tissue-level organized actin bundles, which shorten during contractions. Additionally, a muscle-specific paralog of type II-Myosin heavy chain (stMyHC) is found exclusively at these structures. Contractions appear to be initiated by release of internal Ca2+stores and are regulated by phosphorylation of myosin regulatory light chain by myosin light chain kinase. From a developmental perspective, formation of contractile bundles depends on myocardin related transcription factor (MRTF), an important myogenic factor in many animals. Comparative studies on the myoepithelial muscles of the cnidarian model Nematostella vectensis, found evidence of conserved developmental regulation by MRTF. Taken together, these finding suggest the contractile tissue of E. muelleri shares elements of homology with muscles of other animals and can aid in understanding key cellular innovations involved in the evolution of this tissue type

Smart and Functional Polymers

This book is based on the Special Issue of the journal Molecules on “Smart and Functional Polymers”. The collected research and review articles focus on the synthesis and characterization of advanced functional polymers, polymers with specific structures and performances, current improvements in advanced polymer-based materials for various applications, and the opportunities and challenges in the future. The topics cover the emerging synthesis and characterization technology of smart polymers, core?shell structure polymers, stimuli-responsive polymers, anhydrous electrorheological materials fabricated from conducting polymers, reversible polymerization systems, and biomedical polymers for drug delivery and disease theranostics. In summary, this book provides a comprehensive overview of the latest synthesis approaches, representative structures and performances, and various applications of smart and functional polymers. It will serve as a useful reference for all researchers and readers interested in polymer sciences and technologies

Handbook of Marine Model Organisms in Experimental Biology

"The importance of molecular approaches for comparative biology and the rapid development of new molecular tools is unprecedented. The extraordinary molecular progress belies the need for understanding the development and basic biology of whole organisms. Vigorous international efforts to train the next-generation of experimental biologists must combine both levels – next generation molecular approaches and traditional organismal biology. This book provides cutting-edge chapters regarding the growing list of marine model organisms. Access to and practical advice on these model organisms have become aconditio sine qua non for a modern education of advanced undergraduate students, graduate students and postdocs working on marine model systems. Model organisms are not only tools they are also bridges between fields – from behavior, development and physiology to functional genomics. Key Features Offers deep insights into cutting-edge model system science Provides in-depth overviews of all prominent marine model organisms Illustrates challenging experimental approaches to model system research Serves as a reference book also for next-generation functional genomics applications Fills an urgent need for students Related Titles Jarret, R. L. & K. McCluskey, eds. The Biological Resources of Model Organisms (ISBN 978-1-1382-9461-5) Kim, S.-K. Healthcare Using Marine Organisms (ISBN 978-1-1382-9538-4) Mudher, A. & T. Newman, eds. Drosophila: A Toolbox for the Study of Neurodegenerative Disease (ISBN 978-0-4154-1185-1) Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0)