Browsing by Subject "Protein-protein interactions"
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Item Computational and experimental methods in functional genomics : the good, the bad, and the ugly of systems biology(2008-08) Hart, Glen Traver; Marcotte, Edward M.Seven years into the postgenomic era, we sit atop a mountain of data whose generation was enabled by gene sequencing. The creation, integration, and analysis of these large scale data sets allow us to move forward toward the complementary goals of determining the individual roles of the thousands of uncharacterized mammalian genes and understanding how they work together to produce a healthy human being -- or, perhaps more importantly, how their malfunction results in disease. Collapsing the results of large-scale assays into gene networks provides a useful framework from which we can glean information that advances both of these goals. However, the utility of networks is limited by the quality of the data that goes into them. This study offers seeks to shed some light on the quality and breadth of protein interaction networks, describes a new experimental technique for functional genetic assays in mammalian cell lines, and ultimately suggests a strategy for how to improve the overall utility of the output generated by the systems biology community.Item Electrostatic fields at the functional interface of the protein Ral guanine nucleotide dissociation stimulator determined by vibrational Stark effect spectroscopy(2011-12) Stafford, Amy Jo; Webb, Lauren J.; Anslyn, Eric V.Noncovalent factors, such as shape complementarity and electrostatic driving forces, almost exclusively cause the affinity and specificity for which two or more biological macromolecules organize into a functioning complex. The human oncoprotein p21Ras (Ras) and a structurally identical but functionally distant analog, Rap1A (Rap), exhibit high selectivity and specificity when binding to downstream effector proteins that cannot be explained through structural analysis alone. Both Ras and Rap bind to Ral guanine nucleotide dissociation stimulator (RalGDS) with affinities that differ tenfold instigating diverse cellular functions; it is hypothesized that this specificity of RalGDS to discriminate between GTPases is largely electrostatic in nature. To investigate this hypothesis, electrostatic fields at the binding interface between mutants of RalGDS bound to Rap or Ras are measured using vibrational Stark effect (VSE) spectroscopy, in which spectral shifts of a probe oscillator’s energy is related directly to that probe’s local electrostatic environment and measured by Fourier transform infrared spectroscopy (FTIR). After calibration, the probe is inserted into a known position in RalGDS where it becomes a highly local, sensitive, and directional reporter of fluctuations of the protein’s electrostatic field caused by structural or chemical perturbations of the protein. The thiocyanate (SCN) vibrational spectroscopic probe was systematically incorporated throughout the binding interface of RalGDS. Changes in the absorption energy of the thiocyanate probe upon binding were directly related to the change of the strength of the local electrostatic field in the immediate vicinity of the probe, thereby creating a comprehensive library of the binding interactions between Ras-RalGDS and Rap-RalGDS. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations with the results highlighting the complex structural and electrostatic nature of protein-water interface. Additional SASA studies of the nine RalGDS mutants that bind to Ras or Rap verified that experimentally measured thiocyanate absorption energies are negatively correlated with exposure to water at the protein-water interface. By changing the solvent composition, we confirmed that the cyanocysteine residues that are more exposed to solvent experienced a large difference in absorption energy. These studies reinforce the hypothesis that differences in the electrostatic environment at the binding interfaces of Ras and Rap are responsible for discriminating binding partners.Item Emerging biotechnology to detect weak and/or transient protein-protein interactions(2009-12) Thibodeaux, Gabrielle Nina; Zhang, Zhiwen JonathanProtein-protein interactions are of great importance to a number of essential biological processes including cell cycle regulation, cell-cell interactions, DNA replication, transcription and translation. Thus, an understanding of protein-protein interactions is critical for understanding many facets of cell function. Unfortunately, the tools and methods currently in use to identify and study protein-protein interactions focus largely on high affinity, stable interactions. However, the majority of the protein-protein interactions involved in regulatory processes have weak affinities and are transient in nature. Therefore, it is important to develop new biotechnology capable of detecting weak and/or transient protein-protein interactions in vivo. Here, we describe four new methods that allow for the identification and study of weak and/or transient protein-protein interactions in vivo. First, we developed a rapid method to convert Escherichia coli orthogonal tRNA/synthetase pairs into an orthogonal system for mammalian cells in order to site-specifically incorporate unnatural amino acids into any gene of interest using stop codon suppression. This method will allow the expression and purification of proteins that carry normally transient post-translational modifications. Second, we successfully employed site-specific unnatural amino acid incorporation to chemically cross-link a known homodimer, Sortase A, in vivo. Third, we developed a novel tetracycline repressor-based mammalian two-hybrid system and successfully detected homo- and hetero-dimers that are known to have weak binding constants. Finally, a synthetic antibody (termed a synbody) that binds weakly to the SH3 domain of the proto-oncogene Abelson tyrosine kinase was developed. The synbody can potentially be used as a first generation drug and/or biomarker. We hope that the methods developed in this dissertation will enable the scientific community to better understand weak/transient protein-protein interactions in vivo.Item Formation of nanostructures and weakening of interactions between proteins to design low viscosity dispersions at high concentrations(2014-12) Borwankar, Ameya Umesh; Johnston, Keith P., 1955-; Truskett, Thomas Michael, 1973-; Maynard, Jennifer A; Ganesan, Venkat; Sokolov, Konstantin VMonoclonal antibodies and other protein therapeutics are rapidly gaining popularity as a favored class of drugs for treatment of various types of diseases and disorders including rheumatoid arthritis, Crohn’s disease, asthma, macular degeneration, different types of cancer. There great lot of interest in development of subcutaneous self-injection methods for administering these therapeutics to enable patient convenience which requires high concentration formulations to deliver the required dosage in the limited volume. At high concentrations, proteins have a propensity to be insoluble, aggregate, unfold, gel or denature due to strong short ranged protein-protein interactions, resulting in highly viscous solutions. Therefore, it is challenging to form highly concentrated, stable protein formulations with low viscosities. Addition of interacting co-solutes like arginine to protein formulations weakens protein-protein interactions through protein charge modification and hydrophilization of hydrophobic surface patches through binding on proteins. Weakened interactions lower the viscosity of protein formulations with 250 mg/ml protein by 5-6 times compared to conventional protein solutions in buffer not containing any co-solutes. Addition of co-solutes can also give rise to depletion attraction between proteins which can assemble them into amorphous nanostructured domains with lowered diffusion coefficients as determined by dynamic light scattering (DLS). A free energy model was developed to explain the formation of nanostructures due to short-ranged depletion attraction and long-ranged electrostatic repulsion, whereby sizes were predicted to range from 30 to 100 nm as a function of co-solute and protein concentrations. The nanostructured domains dissociated to monomeric, active and stable protein upon dilution to about 1 mg/ml. Supplemental sizing techniques, namely, cryogenic scanning electron microscopy (cryo-SEM) and small angle x-ray scattering (SAXS) show evidence of nanostructures larger than the monomer although determining the ratio of the amount of protein in monomeric state to that in the nanostructure state is still a challenge. In order to further understand cluster formation in a simpler system, gold nanoclusters were synthesized via assembly of primary particles by reaction. The morphology of these gold nanoclusters was also controlled by favoring kinetic over thermodynamic control of growth for generating asymmetrical structures thus allowing higher extinction in the near infrared region enabling biomedical imaging.Item From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes(2007-12) Li, Zhihua, doctor of cell and molecular biology; Marcotte, Edward M.Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.Item Modeling and visualization of flexible protein-protein interactions(2006) Siddavanahalli, Vinay Kiranshankar; Bajaj, ChandrajitProtein-protein interactions form the basis of macromolecular formation and function. Determining a relative transformation for a pair of proteins and their conformations which form a stable complex, reproducible in nature, is known as protein-protein docking. Computational approaches to proteinprotein docking are therefore a necessary pathway to virtual drug screening, plausible macro-molecular structures, and elucidating the function of proteins in assemblages. Protein conformational changes play a crucial role in such interactions, leading to a very high dimensional search space. The computational challenge is further increased as we obtain imaging data for larger and larger proteins, bridging the gap between proteins and cells. Traditional algorithms for the construction and visualization of protein structure and function have not scaled to handle large proteins, macromolecular assemblies and viruses. In this thesis, we provide: data structures and algorithms to represent flexible protein structures, scalable error bounded techniques to compute soft protein-protein docking, a hierarchical flexible docking scheme and novel methods to visualize large interacting molecular complexes and assemblies. Accurate and robust molecular surface computation is vital for parameterizing affinity functions and modeling interactions. We provide a adaptive grid based function definition, whose contours yield a family of relevant surfaces. We show that these are free of self intersections and provide methods to compute regions of C0 continuity. The structure and functions of molecules are represented in a radial basis format, with smooth particle data representing electron density kernels, charges and solvent modulated dielectric coefficients. A fast summation algorithm, based on non-equispaced fast Fourier transforms, is presented to accurately, efficiently and adaptively compute these functions. Based on the previous surfaces and fast summation algorithms, we provide a model for soft docking and error-bounded approximation algorithms to solve the model and predict docking sites. The flexibility space is adaptively sampled using a domain decomposition of the protein into a Flexible Chain Complex. We then provide a flexible docking algorithm based on a multiresolution representation of the proteins, adaptive sampling of conformation, orientation spaces and greedy fit of residues at interfaces. Scientific visualization of protein interfaces and active sites is employed for both data analysis and discovery. We provide algorithms to interactively render both the traditional ball and stick model of molecules and contours of the sum of Gaussians based electron density. To visualize schematic models of large and flexible proteins at interactive rates and high quality, we introduce a novel hardware accelerated, imposter-based scheme to render curved surfaces like spherical patches, cylinders and helices, with correct per pixel shading, using limited geometric primitives. A telescoping rover is used together with our fast summation algorithm and adaptive isocontouring to efficiently visualize density contours of proteins in a multiresolution fashion. All the above algorithms are implemented in a public domain software package called TexMol.Item Novel tools for the study of protein-protein interactions in pluripotent cells(2011-08) Moncivais, Kathryn Lauren; Zhang, Zhiwen Jonathan; Roy, Krishnendu; Georgiou, George; Ren, Pengyu; Zhang, XiaojingUnnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described.Item Quantifying electrostatic fields at protein interfaces using classical electrostatics calculations(2015-08) Ritchie, Andrew William; Webb, Lauren J.; Elber, Ron; Fast, Walter; Henkelman, Graeme; Ren, PengyuThe functional aspects of proteins are largely dictated by highly selective protein- protein and protein-ligand interactions, even in situations of high structural homology, where electrostatic factors are the major contributors to selectivity. The vibrational Stark effect (VSE) allows us to measure electrostatic fields in complex environments, such as proteins, by the introduction of a vibrational chromophore whose vibrational absorption energy is linearly sensitive to changes in the local electrostatic field. The works presented here seek to computationally quantify electrostatic fields measured via VSE, with the eventual goal of being able to quantitatively predict electrostatic fields, and therefore Stark shifts, for any given protein-interaction. This is done using extensive molecular dynamics in the Amber03 and AMOEBA force fields to generate large ensembles the GTPase Rap1a docked to RalGDS and [superscript p]²¹Ras docked to RalGDS. We discuss how side chain orientations contribute to the differential binding of different mutations of Rap1a binding to RalGDS, where it was found that a hydrogen-bonding pocket is disrupted by the mutation of position 31 from lysine to glutamic acid. We then show that multi-dimensional umbrella sampling of the probe orientations yields a wider range of accessible structures, increasing the quality of the ensembles generated. A large variety of methods for calculating electrostatic fields are presented, with Poisson- Boltzmann electrostatics yielding the most consistent, reliable results. Finally, we explore using AMOEBA for both ensemble-generation as well as the electrostatic description of atoms for field calculations, where early results suggest that the electrostatic field due to the induce dipole moment of the probe is responsible for predicting qualitatively correct Stark shifts.Item A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae: characterization of its interaction with a deubiquitinating enzyme(2006) Kee, Younghoon; Huibregtse, Jon M.HECT E3 ubiquitin ligases are widely distributed from yeast to human cells and play important roles in many physiological processes. Rsp5, an essential HECT E3 ligase in Saccharomyces cerevisiae, is involved in many biological processes, including transcriptional activation, endocytic trafficking, mitochondrial inheritance, and RNA export pathways. Although Rsp5 has been shown to regulate multiple pathways targeting multiple substrates, mechanisms for regulating the biochemical activity of Rsp5 are largely uncharacterized (121, 199). To gain further insight into the regulation of this enzyme, I identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Biochemical and genetic evidence are consistent with a model that Ubp2and Rup1 antagonizes Rsp5-catalyzed substrate ubiquitination. In vivo and in vitro experiments showed that Rsp5 and Ubp2 display strong preferences for assembly and disassembly of K63-linked polyubiquitination, respectively. A large fraction of the K63 conjugates in ubp2∆ cells bound to Rsp5, and a proteomics approach was therefore used to identify Rsp5 substrates subject to Ubp2 regulation. Two proteins implicated in cell wall integrity, Csr2 and Ecm21, were identified and both proteins were efficiently K63- polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. I have also shown that cell wall integrity is impaired in rsp5-1 cells and this can be rescued by either ubp2∆ or rup1∆ mutation, suggesting that the Ubp2/Rup1 complex negatively regulates Rsp5-mediated cell wall homeostasis. Together, these data represent a novel regulatory mechanism for Rsp5 and suggest that similar mechanisms might be utilized by its mammalian homologues. Furthermore, this work provides a basis for studying the mechanism for differential polyubiquitin chain type synthesis by HECT E3 ligases.Item Role of local electrostatic fields in protein-protein and protein-solvent interactions determined by vibrational Stark effect spectroscopy(2014-05) Ragain, Christina Marie; Webb, Lauren J.This examines the interplay of structure and local electrostatic fields in protein-protein and protein-solvent interactions. The partial charges of the protein amino acids and the polarization of the surrounding solvent create a complex system of electrostatic fields at protein-protein and protein-solvent interfaces. An approach incorporating vibrational Stark effect (VSE) spectroscopy, dissociation constant measurements, and molecular dynamics (MD) simulations was used to investigate the electrostatic interactions in these interfaces. Proteins p21Ras (Ras) and Rap1A (Rap) have nearly identical amino acid sequences and structures along the effector-binding region but bind with different affinities to Ral guanine nucleotide dissociation stimulator (RalGDS). A charge reversion mutation at position 31 alters the binding affinity of Ras and Rap with RalGDS from 0.1 [mu]M and 1 [mu]M, to 1 [mu]M and 0.5 [mu]M, respectively. A spectral probe was placed at various locations along the binding interface on the surface of RalGDS as it was docked with Ras and Rap single (position 30 or 31) and double mutants (both positions). By comparing the probes' absorption energies with the respective wild-type (WT) analogs, VSE spectroscopy was able to measure molecular-level electrostatic events across the protein-protein interface. MD simulations provided a basis for deconvoluting the structural and electrostatic changes observed by the probes. The mutation at position 31 was found to be responsible for both structural and electrostatic changes compared to the WT analogs. Furthermore, previous identification of positions N27 and N29 on RalGDS as "hot spots" that help discriminate between structurally similar GTPases was supported. The RalGDS probe-containing variants and three model compounds were placed in aqueous solvents with varying dielectric constants to measure changes in absorption energy. We investigated the ability of the Onsager solvent model to describe the solvent induced changes in absorption energy, while MD simulations were employed to determine the location and solvation of the probes at the protein-solvent interface. The solvent accessible-surface area, a measure of hydration, was determined to correlate well with the change in magnitude of the probe's absorption energy and the displaced solvent by the probe.Item Studies in pharmaceutical biotechnology : protein-protein interactions and beyond(2011-05) Umeda, Aiko; Zhang, Zhiwen Jonathan; Georgiou, George; Liu, Hung-wen; Johnson, Kenneth A.; Browning, Karen S.Pharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools.