The Fuzziness in Molecular, Supramolecular, and Systems Chemistry

Fuzzy Logic is a good model for the human ability to compute words. It is based on the theory of fuzzy set. A fuzzy set is different from a classical set because it breaks the Law of the Excluded Middle. In fact, an item may belong to a fuzzy set and its complement at the same time and with the same or different degree of membership. The degree of membership of an item in a fuzzy set can be any real number included between 0 and 1. This property enables us to deal with all those statements of which truths are a matter of degree. Fuzzy logic plays a relevant role in the field of Artificial Intelligence because it enables decision-making in complex situations, where there are many intertwined variables involved. Traditionally, fuzzy logic is implemented through software on a computer or, even better, through analog electronic circuits. Recently, the idea of using molecules and chemical reactions to process fuzzy logic has been promoted. In fact, the molecular word is fuzzy in its essence. The overlapping of quantum states, on the one hand, and the conformational heterogeneity of large molecules, on the other, enable context-specific functions to emerge in response to changing environmental conditions. Moreover, analog input–output relationships, involving not only electrical but also other physical and chemical variables can be exploited to build fuzzy logic systems. The development of “fuzzy chemical systems” is tracing a new path in the field of artificial intelligence. This new path shows that artificially intelligent systems can be implemented not only through software and electronic circuits but also through solutions of properly chosen chemical compounds. The design of chemical artificial intelligent systems and chemical robots promises to have a significant impact on science, medicine, economy, security, and wellbeing. Therefore, it is my great pleasure to announce a Special Issue of Molecules entitled “The Fuzziness in Molecular, Supramolecular, and Systems Chemistry.” All researchers who experience the Fuzziness of the molecular world or use Fuzzy logic to understand Chemical Complex Systems will be interested in this book

Bifunktsionaalsed inhibiitorid ja fotoluminestsents-sondid valgukomplekside uurimiseks

Väitekirja elektrooniline versioon ei sisalda publikatsiooneInimraku elutegevus on reguleeritud keeruliste protsesside kaudu. Ühed kõige olulisemad informatsiooni edasikandvad muutused on valkudevahelised interaktsioonid ja valkude fosforüülimine. Valkude fosforüülimist teostavad proteiinkinaasid, mis katalüüsivad fosforüülrühma ülekannet nukleotiidilt (milleks on enamasti ATP) sihtvalgule. Kõrvalekaldeid proteiinkinaaside normaalsest aktiivsuses on seostatud komplekssete ja sageli raskesti ravitavate haigustega, näiteks erinevad neurodegeneratiivsed haigused (Parkinson, Alzheimer), südame- ja veresoonkonnahaigused, diabeet ning vähkkasvajad. Seetõttu on ravimitööstuse kõrgendatud tähelepanu suunatud proteiinkinaaside aktiivsuse reguleerimisele inhibiitoritega. Tänaseks päevaks on kasutusloa saanud enam kui 30 proteiinkinaaside inhibiitorit, mis on märkimisväärselt aidanud parandada vähktõbe põdevate inimesete elukvaliteeti ja elulemust. Käesoleva töö raames töötati välja ja iseloomustati mitmekülgseid ja tundlikke meetodeid, mis võimaldasid iseloomustada mitmete proteiinkinaaside aktiivsust biokeemilistes katsetes, inhibiitorite sidumise tugevust proteiinkinaasidele, proteiinkinaas:inhibiitor kompleksi lagunemise kiirust ja jälgida reaalajas proteiinkinaasi aktiivsust imetajarakkudes. Eriliseks teeb meetodid nendes rakendatud orgaaniline sond ARC-Lum, millel on unikaalsed optilised omadused. Esiteks, proovi ergastamisel kiirgub valgust vaid juhul, kui ARC-Lum sond on kompleksis proteiinkinaasiga. Teiseks, kiirgunud valgusel on pikk eluiga, mis võimaldab vähendada mittespetsiifilise signaali mõjusid. ARC-Lum sondi rakendamisel arendati uudse struktuuriga bifunktsionaalsed ARC-tüüpi inhibiitorid, mis saavutasid senikirjeldatud inhibiitoritest kõrgeima afiinsuse proteiinkinaasi PKAc suhtes (Kd < 10 pM). Võrreldes ravimina kasutatavate inhibiitoritega inhibeerivad uued ained proteiinkinaase oluliselt (kuni tuhat korda) madalamatel kontsentratsioonidel. Arendatud inhibiitorite võimekust demonstreeriti väga tugeva valkudevahelise interaktsiooni lõhkumisel ja pakuti välja uudne lahendus sihtida tugevaid valkudevahelisi interaktsioone bifunktsionaalsete inhibiitoritegaA living cell is highly complex and dynamic system, regulated by various signaling pathways. Interactions between proteins and phosphorylation of proteins are two of the most important changes in cells, carrying important information. The phosphorylation of proteins is aided by protein kinases, which catalyze the transfer of phosphoryl group from nucleotide (usually ATP) to target protein. Abnormal activity of protein kinases has been linked to complex and refractory diseases, for example different neurodegenerative diseases (Parkinson’s, Alzheimer’s), cardiovascular diseases, diabetes, and cancer. Therefore, pharmaceutical industry has directed considerable effort into regulating the activity of protein kinases with inhibitors. Currently 30 protein kinase inhibitors have been approved for therapeutics against various forms of cancer, immensely improving the life expectancy and quality of living of patients. In the current thesis, versatile and sensitive methods were developed and characterized for studying the activity of protein kinases in biochemical assays, determining interaction strength of inhibitors towards protein kinases, measuring dissociation kinetics of a complex between protein kinase and inhibitor, and monitoring cellular activity of protein kinases in real time. The novelty of these methods is based on organic ARC-Lum probes with unique optical properties. First, the specific emission of light is present only if ARC-Lum is in complex with protein kinase. Second, emitted light has a characteristic long lifetime, which allows to reduce the influence from non-specific signals. These properties of ARC-Lum probes supported the development of novel bifunctional ARC-type inhibitors with the highest affinity towards protein kinase PKAc to date (Kd < 10 pM). Compared to inhibitors in drug industry, these new compounds inhibit protein kinases at significantly (up to 1000-fold) lower concentrations. These potent inhibitors performed exceedingly well for disruption of a very strong protein- protein interaction (PPI) and led to a proposal of targeting strong PPIs by using bifunctional inhibitors

Calcium Regulation of Cell-Cell Communication and Extracellular Signaling

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Molecular Basis Of Bar Domain Super-Family Proteins And Genetically Encoded Calcium Indicators

Protein domains are the basic functional modules that maintain cell functions at a molecule level. Previous studies have mainly focused on the functions of isolated protein domains. The general objective of this thesis is to understand functions and regulations of multi-domain containing proteins. The study is based on two protein classes: naturally occurred BAR domain-containing proteins and artificially engineered calcium indicators. BAR domain super-family proteins BAR (Bin/Amphiphysin/Rvs) domain super-family proteins are peripheral membrane proteins that regulate membrane curvatures during the membrane remodeling events such as endocytosis, vesicular trafficking and cell growth. Via multiple biophysical approaches, I systematically studied BAR domain functions in Sorting Nexin 9, Endophilin and Pacsins at the presence of other protein domains. Two major findings are presented in this thesis. First I show that the diverse membrane sculpture activity of BAR domains is encoded in their unique molecular structures, and is influenced by membrane properties. Second, I show that this function diversity is highly regulated by other protein domains. Some protein domains have synergetic effects and play important roles in regulating cellular membrane remodeling. This work is significant in that it provides the molecular basis for the functional diversity of BAR domains and established the regulatory mechanism of BAR domain mediated-membrane deformation process. Genetically Encoded Calcium Indicators Genetically encoded calcium indicator GCAMP is an artificially designed multi-domain containing protein that can be endogenously expressed in cells to monitor calcium signals. The molecular mechanisms of its signal response and fast kinetics are poorly understood. Using fluorescent spectrometry and site-directed mutagenesis, I show that the calcium-dependent brightness of GCAMP is due to the different protonation states of the chromophore. Structural characterization of GCAMP reveals that the calmodulin domain regulates chromophore protonation states via a sophisticated water-mediated hydrogen bond network. This finding provided a general scheme for designing GCAMP-like sensors. Furthermore, I show that distinct electron properties of the protonated and deprotonated chromophore can be applied to design color switchable fluorescent proteins. This finding provides a novel approach to design the ratio metric pH sensors with an improved sensitivity

52nd Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 52nd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Snowmass, Colorado, August 1-5, 2010

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Investigating the role of orphan GPR50 in normal brain function and mental illness

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signaling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. G protein-coupled receptor 50 (GPR50) is one such orphan, and its exact role is yet unknown. There is however emerging functional and genetic evidence suggesting a function for GPR50 in psychiatric illness and lipid metabolism. It was hypothesised that investigating GPR50’s protein-protein interactions would lead to a greater understanding of the role of GPR50 in normal brain functioning and in mental illness. Putative protein interactors were initially isolated by a yeast two-hybid study and were further tested here. To address GPR50’s links to mental illness, the GPR50∆502-505 deletion variant associated with mood disorders was also investigated. To test this hypothesis I sought to confirm some of the key yeast two-hybrid interactions. Using co-immunoprecipitation and immunocytochemistry the interaction of GPR50 with reticulon family members Nogo-A, Nogo-C and RTN3, and with cell-cell adhesion molecule CDH8 and lipid-associated protein ABCA2 were validated. In order to identify the location of interactions, subcellular fractionation of mouse brain and rt-PCR and immunohistochemistry in developing and adult mouse brain were performed. GPR50 and several interactors were found to be enriched at the synapse by subcellular fractionation of whole adult brain, and at embryonic day 18 (E18) and 5 weeks by rt-PCR. Colocalisation of GPR50 and interactors was found in the amygdala, hypothalamus, cortex and specific brain stem nuclei by immunohistochemistry. The discovery of GPR50 expression in noradrenergic, serotonergic and dopaminergic nuclei in the adult brain stem suggests a further role for GPR50 in neurotransmitter signaling and stress. To investigate the function of GPR50 two assays were performed that measure processes which are known to be affected by Nogo and RTN3: The first assay was a neurite outgrowth assay in Neuroscreen-1 cells, a PC12 cell clone. A significant increase in neurite length was detected after transient overexpression of GPR50 and this effect was increased in the GPR50∆502-505/T532A variant. Additionally GPR50-overexpression resulted in an increase in filopodia formation suggesting a role in actin dynamics. As a second functional assay in vitro BACE1 activity assays were performed in HEK293 cells. GPR50 but not GPR50∆502-505/T532A overexpression resulted in a significant increase in BACE1 activity. Lastly a final series of pilot experiments were performed to gain insight into the secondary structure of the C-terminal domain and the effects of the polymorphisms on structure. The 35kDa GPR50 C-terminal domain was purified and Circular Dichroism studies indicated a predominantly unstructured protein with increased a- helical content in the GPR50∆502-505 variant. The results in this thesis indicate a role for GPR50 in neuronal development and synaptic functioning. The results also strengthen an association with major mental illness, with links to several disease mechanisms