<p dir="ltr">Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here we described two methods for the LC-MS/MS-based characterization of protein phosphatases and kinases. The first is an <i>in-vitro</i> method designed to probe the inherent substrate specificity of kinase or phosphatases. This method utilizes an enzyme reaction with synthetic peptides, serving served as substrate proxies, coupled with LC-MS/MS for rapid, accurate high-throughput quantification of the specificity constant (<i>k</i>_<em>cat</em><i>/K</i>_<em>M</em>) for each substrate in the reaction and amino acid preference in the enzyme active site, providing insight into their cellular roles. The second couple’s auxin-inducible degradation system (AID) with phosphoproteomics for protein functional characterization. AID is a surrogate for specific chemical inhibition, which minimizes non-specific effects associated with long-term target perturbation. Using this system, we demonstrate-PP2A in complex with its B-subunit Rox Three Suppressor 1 (PP2A^Rts1) contributes to the phosphoregulation of a conserved fungal-specific membrane protein complex called the eisosome. By maintaining eisosomes in their hypophosphorylated state, PP2A^Rts1 aids fungal cells in preserving metabolic homeostasis. This work demonstrates the power of mass spectrometry as a critical tool for protein functional characterization.</p>

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<p> The striatum is a key brain region involved in regulating motor output and integration. The dorsal and ventral subdivisions of the striatum work in concert to mediate the reinforcing and motor behavioral outputs of the striatum. Moreover, dysfunction of these striatal regions is involved in various diseases including Parkinson’s disease and drug addiction. Therefore, understanding and characterizing biochemical and molecular changes within the striatum associated with these diseases is key in devolving novel therapeutics to treat these disease states. The main output neurons of the striatum are GABAergic, medium-spiny neurons (MSNs), and striatal functionality is mediated by neuroplastic changes in MSN activity. Within MSNs, dopaminergic receptor activation triggers a cascade of reversable phosphorylation, which is facilitated by the activation of specific protein kinases and inhibition of specific protein phosphatases. In comparison to the 350 serine/threonine protein kinases expressed within the striatum, there are only 40 major serine/threonine protein phosphatases. However, serine/threonine protein phosphatases, such as protein phosphatase 1 (PP1), gain their target specificity by interacting with phosphatase-targeting proteins. Within the striatum, the neurabins, termed neurabin and spinophilin, are the most abundant PP1 targeting proteins in dendritic spines. Spinophilin’s expression in the striatum has been strongly characterized, and spinophilin has been shown to regulate striatal-dependent motor-skill learning and amphetamine-induced locomotor sensitization. In contrast to spinophilin, neurabin’s expression within the striatum and its involvement in these striatal-dependent behaviors has not been fully probed. I found that neurabin expression in the striatum is not sex-dependent but is age-dependent. In addition to these data, I also present validation of new global, constitutive and conditional neurabin knock-out mouse lines. Finally, I present data that, unlike previous studies in spinophilin knockout mice, neurabin knockout mice have enhanced striatal-dependent motor-skill learning, but do not impact amphetamine-induced locomotor sensitization. Further characterization of neurabin’s expression in the striatum, and its role in these key striatal behaviors could provide a druggable target for therapeutics designed to address striatal dysfunction.   </p>

<p>Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer’s disease (AD) and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, I apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry (FCMS), to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, I detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, 15 candidate kinases were successfully expressed and purified, and four candidate kinases, OXSR1, DAPK2, CSK, and ZAP70, displayed the ability to phosphorylate Tau. Furthermore, co-expression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. I demonstrate that FCMS is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing AD therapeutic strategies.</p>

No / We investigated five different methods for evaluating the electrostatic interaction between atoms in force field calculations on organic solids. Atomic charges and multipoles were obtained by fitting them to the molecular electrostatic potential, calculated in vacuum with an ab initio quantum mechanical method. Multipole moments were derived using three schemes, differing in the order in which the monopoles, dipoles and quadrupoles were fitted. For comparison, Gasteiger charges were also calculated. Using these electrostatic models, the lattice parameters and the molecular geometry of 48 organic crystals were optimised with the DREIDING force field. During the optimisation, the atomic multipoles were rotated with their local environment to account for molecular flexibility. For comparative reasons, rigid-body optimisations were performed on a subset of structures. The results were analysed in terms of structural parameters of the lattice and the molecules, and, for the ten polymorphic systems present in the test set, in terms of relative stability. On average, the multipole methods were not superior to the point charge methods for the full optimisation. For rigid molecules, however, the multipole models gave a substantial improvement in lattice parameters. No evidence was found that parameters for van der Waals energies need to be refitted for a specific electrostatic model. Energy differences between polymorphs were less than 5 kcal mol¿1 in eight out of ten cases, independent of the electrostatic model used. The results show that our use of distributed multipoles to describe the intra-molecular as well as inter-molecular electrostatic interactions does lead to an improvement in accuracy for rigid molecules, but not for flexible molecules. The investigation shows that accurate descriptions of the intra-molecular as well as the inter-molecular energies are crucial for the successful optimisation of crystal structures of organic solids.

Yes / The creation of co-crystals as a route to creating new pharmaceutical phases with modified or defined physicochemical properties is an area of intense research. Much of the current research has focused on creating new phases for numerous active pharmaceutical ingredients (APIs) to alter physical properties such as low solubilities, enhancing processability or stability. Such studies have identified suitable co-formers and common bonding motifs to aid with the design of new co-crystals but understanding how the changes in the molecular structure of the components are reflected in the packing and resulting properties is still lacking. This lack of insight means that the design and growth of new co-crystals is still a largely empirical process with co-formers selected and then attempts to grow the different materials undertaken to evaluate the resulting properties. This work will report on the results of a combination of crystal structure database analysis with computational chemistry studies to identify what structural features are retained across a selection of families of co-crystals with common components. The competition between different potential hydrogen bonding motifs was evaluated using ab initio quantum mechanical calculations and this was related to the commonality in the packing motifs when observed. It is found while the stronger local bonding motifs are often retained within systems, the balance of weaker long-range packing forces gives rise to many subtle shifts in packing leading to greater challenges in the prediction of final crystal structures.

Weak intermolecular interactions have very strong impact on the structures and properties of life giving molecules like H2O, DNA, RNA etc. These interactions are responsible for many biological phenomena. The directional preference of some of these interactions is used for designing different synthetic approaches in the supramolecular chemistry. The work reported in this Thesis comprises of investigations of weak intermolecular interactions in gas phase using home-built Pulsed Nozzle Fourier Transform Microwave (PN-FTMW) spectrometer as an experimental tool and ab-initio and Atoms in Molecules (AIM) theory as theoretical tools. The spectrometer which is coupled with a pulsed nozzle is used to record pure rotational spectra of the molecular clusters in a jet cooled molecular beam. In the molecular beam molecules/complexes are free from interactions with other molecules/complexes and thus, spectroscopy in the molecular beams provides information about the 'isolated' molecule/complex under investigation. The rotational spectra of the molecules/complexes in the molecular beam provide their geometry in the ground vibrational states. These experimental geometries can be used to test the performance and accuracy of theoretical models like ab-initio theory, when applied to the weakly bound complexes. Further the AIM theory can be used to gain insights into the nature and strength of the intermolecular interactions present in the system under investigation. Chapter I of this Thesis gives a brief introduction of intermolecular interactions. Other than hydrogen bonding, which is considered as the most important intermolecular interaction, many other intermolecular interactions involving different atoms have been observed in past few decades. The chapter summarizes all these interactions. The chapter also gives a brief introduction to the experimental and theoretical methods used to probe these interactions. In Chapter II, the experimental and theoretical methods used in this work are summarized. Details of our home-built PN-FTMW spectrometer are given in this chapter. The chapter also discusses briefly the theoretical methods like ab-initio, AIM and Natural bond orbital (NBO) analysis. We have made few changes in the mode of control of one of our delay generators which have also been described. Chapter III and Chapter V of this Thesis are dedicated to the propargyl alcohol complexes. Propargyl alcohol (PA) is a molecule of astrophysical interest. It is also important in combustion chemistry since propargyl radical is considered as the precursor in soot formation. Moreover, PA is a multifunctional molecule, having a hydroxyl (-OH) and an acetylenic (-C≡C-H) group. Both of the groups can individually act as hydrogen bond acceptor as well as donor and thus PA provides an exciting possibility of studying many different types of weak interactions. Due to internal motion of -OH group, PA monomer can exist in gauche as well as trans form. However, rotational spectra of PA-monomer show the presence of only gauche conformer. In Chapter III, rotational spectra of Ar•••PA complex are discussed. The pure rotational spectra of the parent Ar•••PA complex and its two deuterated isotopologues, Ar•••PA-D (OD species) and Ar•••PA-D (CD species), could be observed and fitted within experimental uncertainty. The structural fitting confirmed a structure in which PA is present as gauche conformer and argon interacts with both the O-H group and the acetylenic group leading to Ar•••H-O and Ar•••π interactions respectively. Presence of these interactions was further confirmed by AIM theoretical analysis. In all the three isotopologues c-type rotational transitions showed significant splitting. Splitting patterns in the three isotopologues suggest that it originates mainly due to the large amplitude motion of the hydroxyl group and the motion is weakly coupled with the carbon chain bending motion. No evidence for the complex with trans conformer of PA was found. Although, we could not observe Ar•••trans-PA complex experimentally, we decided to perform ab-initio and AIM theoretical calculations on this complex as well. AIM calculations suggested the presence of Ar•••H-O and a unique Ar•••C interaction in this complex which was later found to be present in the Ar•••methanol complex as well. This prompted us to explore different possible interactions in methanol, other than the well known O-H•••O hydrogen bonding interactions, and eventually led us to an interesting interaction which we termed as carbon bond. Chapter IV discusses carbon bonding interaction in different complexes. Electrostatic potential (ESP) calculations show that tetrahedral face of methane is electron-rich and thus can act as hydrogen/halogen bond acceptor. This has already been observed in many complexes, e.g. CH4•••H2O/HF/HCl/ClF etc., both experimentally and theoretically. However, substitution of one of the hydrogens of methane with -OH leads to complete reversal of the properties of the CH3 tetrahedral face and this face in methanol is electron-deficient. We found that CH3 face in methanol interacts with electron rich sites of HnY molecules and leads to the formation of complexes stabilized by Y•••C-X interactions. This interaction was also found to be present in the complexes of many different CH3X (X=OH/F/Cl/Br/NO2/NF2 etc.) molecules. AIM, NBO and C-X frequency shift analyses suggest that this interaction could be termed as "carbon bond". The carbon bonding interactions could be important in understanding hydrophobic interactions and thus could play an important role in biological phenomena like protein folding. The carbon bonding interaction could also play a significant role in the stabilization of the transition state in SN2 reactions. In Chapter V of this Thesis rotational spectra of propargyl alcohol dimer are discussed. Rotational spectra of the parent dimer and its three deuterated (O-D) isotopologues (two mono-substituted and one bi-substituted) could be recorded and fitted within experimental uncertainty. The fitted rotational constants are close to one of the ab-initio predicted structure. In the dimer also propargyl alcohol exists in the gauche form. Atoms in molecules analysis suggests that the experimentally observed dimer is bound by O-H•••O, O-H•••π and C-H•••π interactions. Chapter VI of the thesis explores the 'electrophore concept'. To observe the rotational spectra of any species and determine its rotational constant by microwave spectroscopy, the species should have a permanent dipole moment. Can we obtain rotational constants of a species having no dipole moment via microwave spectroscopy? Electrophore concept can be used for this purpose. An electrophore is an atom or molecule which could combine with another molecule having no dipole moment thereby forming a complex with a dipole moment, e.g. Argon atom is an electrophore in Ar•••C6H6 complex. The microwave spectra of Ar•••13CC5H6 and Ar•••C6H5D complexes were recorded and fitted. The A rotational constant of these complexes was found to be equal to the C rotational constant of 13CC5H6 and C6H5D molecules respectively and thus we could determine the C rotational constant of microwave 'inactive' 13CC5H6. This concept could be used to obtain the rotational spectra of parallel displaced benzene-dimer if it exists. We recently showed that the square pyramidal Fe(CO)5 can act as hydrogen bond acceptor. Appendix I summarizes the extension of this work and discusses interactions of trigonal bipyramidal Fe(CO)5 with HF, HCl, HBr and ClF. Our initial attempts on generating a chirped pulse to be used in a new broadband spectrometer are summarized in Appendix II. Preliminary investigations on the propargyl•••water complex are summarized in Appendix III.

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