Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding

10.1186/1471-2105-14-S16-S11BMC Bioinformatics14SUPPL16-BBMI

Protein Ligand Interactions Probed by NMR: A Dissertation

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease

Molecular Dynamics Investigations of Structural Conversions in Transformer Proteins

Multifunctional proteins that undergo major structural changes to perform different functions are known as “Transformer Proteins”, which is a recently identified class of proteins. One such protein that shows a remarkable structural plasticity and has two distinct functions is the transcription antiterminator, RfaH. Depending on the interactions between its N-terminal domain and its C-terminal domain, the RfaH CTD exists as either an all-α-helix bundle or all-β-barrel structure. Another example of a transformer protein is the Ebola virus protein VP40 (eVP40), which exists in different conformations and oligomeric states (dimer, hexamer, and octamer), depending on the required function.I performed Molecular Dynamics (MD) computations to investigate the structural conversion of RfaH-CTD from its all-a to all-b form. I used various structural and statistical mechanics tools to identify important residues involved in controlling the conformational changes. In the full-length RfaH, the interdomain interactions were found to present the major barrier in the structural conversion of RfaH-CTD from all-a to all-b form. I mapped the energy landscape for the conformational changes by calculating the potential of mean force using the Adaptive Biasing Force and Jarzynski Equality methods. Similarly, the interdomain salt-bridges in the eVP40 protomer were found to play a critical role in domain association and plasma membrane (PM) assembly. This molecular dynamic simulation study is supported by virus like particle budding assays investigated by using live cell imaging that highlighted the important role of these saltbridges. I also investigated the plasma membrane association of the eVP40 dimer in various PM compositions and found that the eVP40 dimer readily associates with the PM containing POPS and PIP2 lipids. Also, the CTD helices were observed to be important in stabilizing the dimer-membrane complex. Coarse-grained MD simulations of the eVP40 hexamer and PM system revealed that the hexamer enhances the PIP2 lipid clustering at the lower leaflet of the PM. These results provide insight on the critical steps in the Ebola virus life cycle

Current and Novel Inhibitors of HIV Protease

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed

Transmembrane Domain Structure and Function in the Erythropoietin Receptor

a dissertatio

Dynamics of allosteric regulation of the phospholipase C-gamma isozymes

The phospholipase C (PLC)-γ isozymes (-γ1 and -γ2) are activated by phosphorylation downstream of numerous tyrosine kinases including receptor tyrosine kinases for growth factors. Once phosphorylated, PLC-γ isozymes catalyze the hydrolysis of the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers 1,2-diacyglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The regulated activation of this signaling pathway controls diverse biological processes including vasculature, chemotaxis, and immunity. Conversely, aberrant PLC-γ isozyme signaling leads to human diseases including cancer and immune disorders. The work presented in this dissertation describes a protocol for the quantification of PLC activity in vitro, the crystal structure of autoinhibited PLC-γ1, and a model of allosteric control of PLC-γ isozyme activation based on hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). First, we developed a protocol for the quantification of PLC activity in vitro using a fluorogenic substrate that partitions into membranes. This assay permits the continuous monitoring of PLC activity at a pH of 7.4 ± 0.2 and at a concentration of free calcium of approximately 400 nM. This assay can be modified to include small molecules, peptides, and effectors. Second, we describe the crystal structure of PLC-γ1 in the autoinhibited state. The structure illustrates the arrangement of the regulatory domains relative to the catalytic core to form two interfaces that effectively block access to the active site. Simultaneously, the regulatory domains are arranged to integrate inputs from adaptor and signaling proteins. Finally, the dynamics of PLC-γ isozyme activation were assessed with a 1:1 complex of PLC-γ1 bound to phosphorylated kinase domain of the fibroblast growth factor receptor 1 (FGFR1K). According to HDX-MS, binding of PLC-γ1 to the kinase causes widespread changes in deuterium incorporation that suggest a loosening of the interfaces between the regulatory domains and the catalytic core. These results suggest that kinase engagement shifts the dynamic equilibrium of PLC-γ isozymes from an autoinhibited conformation to a more active conformation. In addition, oncogenic substitution of PLC-γ1 mimics the effects of kinase engagement and uncovers functional cooperativity of kinases and lipid bilayers.Doctor of Philosoph

Role of noncovalent interactions in protein peripheral membrane binding. Computational perspectives

Noncovalent forces are important driving forces in nature particularly in biology, and they dictate many biological processes including the binding of peripheral protein to the cell membrane. The widely acknowledged models describe this process as electrostatics driven membrane adsorption followed by short-range protein-lipid interactions i.e. hydrogen bonds, hydrophobic interactions. Some of the key elements in such models are: clusters of basic residues are essential for electrostatic adsorption, and basic residues contribute equally to the membrane binding. Nevertheless, none of these models account for the role of cation-π interactions in membrane binding. With selected protein candidates, we further explore these models and work towards a generalized description of protein peripheral binding to membranes in terms of noncovalent forces. Our investigation highlights the limitations of these existing descriptions. We demonstrate that the requirement of having a cluster of basic residues is not essential. Further, we show that the contributions of basic residues are distance dependent. In other words, their localization in the membrane-water interface determines their strength and hence is not equal. We also establish the role of tyrosine-choline cation- π interactions in membrane binding of peripheral proteins. We explore in detail the nature of tyrosine-choline mediated cation-π interactions using high-level quantum mechanical calculations. Later, this information is used to improve the description of cation-π interactions in molecular simulation models. These improvements of force field parameters are further tested using molecular dynamics simulations. Finally, we used this information to build an interaction diagram that can be used to better describe the binding of peripheral proteins to the cell membrane. Future testing and the generalization of this diagram will further establish this as a common model