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Algorithmic Prediction of Binding Sites of TNFα/TNFR2 and PD-1/PD-L1

Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of…

Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Computational Study of Conformation Dynamics and Allostery in WW Protein Domains

Description
Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46…

Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46 residue-long, protein interaction module composed of a WW domain and corresponding polyproline ligand (PDB id: 1k9r). The dynamic flexibility index (DFI) was computed for the binding site of the wild type and mutant WW domains to quantify the mutations effect on the rigidity of the binding pocket. DFI is used as a metric to quantify the resilience of a given position to perturbation along the chain. Using steered molecular dynamics (SMD), we also measure the effect of the point mutation on allosteric regulation by approximating the binding free energy of the system calculated using Jarzynski's Equality. Calculation of the DFI shows that the overall flexibility of the protein complex increases as a result of the distal point mutation. Total change in DFI percentile of the binding site showed a 0.067 increase suggesting an allosteric, loss of function mutation. Furthermore, we see that the change in the binding free energy is greater for that of the mutated complex supporting the idea that an increase in flexibility is correlated to a decrease in proteinlig and binding affinity. We show that sequence mutation of an allosteric site affects the mechanical stability and functionality of the binding pocket.
ContributorsMarianchuk, Tegan (Author) / Ozkan, Sefika (Thesis director) / Ros, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor)
Created2018-05
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Entanglement, Locality, and Hidden Variables

Description

The purpose of this paper is to provide an analysis of entanglement and the particular problems it poses for some physicists. In addition to looking at the history of entanglement and non-locality, this paper will use the Bell Test as a means for demonstrating how entanglement works, which measures the…

The purpose of this paper is to provide an analysis of entanglement and the particular problems it poses for some physicists. In addition to looking at the history of entanglement and non-locality, this paper will use the Bell Test as a means for demonstrating how entanglement works, which measures the behavior of electrons whose combined internal angular momentum is zero. This paper will go over Dr. Bell's famous inequality, which shows why the process of entanglement cannot be explained by traditional means of local processes. Entanglement will be viewed initially through the Copenhagen Interpretation, but this paper will also look at two particular models of quantum mechanics, de-Broglie Bohm theory and Everett's Many-Worlds Interpretation, and observe how they explain the behavior of spin and entangled particles compared to the Copenhagen Interpretation.
ContributorsWood, Keaten Lawrence (Author) / Foy, Joseph (Thesis director) / Hines, Taylor (Committee member) / Department of Physics (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Purification of the P66 Outer Membrane Protein of the Bacterium Borrelia burgdorferi

Description

Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target…

Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Evolutionary Guided Molecular Dynamics Driven Protein Design

Description
Natures hardworking machines, proteins, are dynamic beings. Comprehending the role of dynamics in mediating allosteric effects is paramount to unraveling the intricate mechanisms underlying protein function and devising effective protein design strategies. Thus, the essential objective of this thesis is to elucidate ways to use protein dynamics based tools integrated…

Natures hardworking machines, proteins, are dynamic beings. Comprehending the role of dynamics in mediating allosteric effects is paramount to unraveling the intricate mechanisms underlying protein function and devising effective protein design strategies. Thus, the essential objective of this thesis is to elucidate ways to use protein dynamics based tools integrated with evolution and docking techniques to investigate the effect of distal allosteric mutations on protein function and further rationally design proteins. To this end, I first employed molecular dynamics (MD) simulations, Dynamic Flexibility Index (DFI) and Dynamic Coupling Index (DCI) on PICK1 PDZ, Butyrylcholinesterase (BChE), and Dihydrofolate reductase (DHFR) to uncover how these proteins utilize allostery to tune activity. Moreover, a new classification technique (“Controller”/“Controlled”) based on asymmetry in dynamic coupling is developed and applied to DHFR to elucidate the effect of allosteric mutations on enzyme activity. Subsequently, an MD driven dynamics design approach is applied on TEM-1 β-lactamase to tailor its activity against β-lactam antibiotics. New variants were created, and using a novel analytical approach called "dynamic distance analysis" (DDA) the degree of dynamic similarity between these variants were quantified. The experimentally confirmed results of these studies showed that the implementation of MD driven dynamics design holds significant potential for generating variants that can effectively modulate activity and stability. Finally, I introduced an evolutionary guided molecular dynamics driven protein design approach, integrated co-evolution and dynamic coupling (ICDC), to identify distal residues that modulate binding site dynamics through allosteric mechanisms. After validating the accuracy of ICDC with a complete mutational data set of β-lactamase, I applied it to Cyanovirin-N (CV-N) to identify allosteric positions and mutations that can modulate binding affinity. To further investigate the impact of mutations on the identified allosteric sites, I subjected putative mutants to binding analysis using Adaptive BP-Dock. Experimental validation of the computational predictions demonstrated the efficacy of integrating MD, DFI, DCI, and evolution to guide protein design. Ultimately, the research presented in this thesis demonstrates the effectiveness of using evolutionary guided molecular dynamics driven design alongside protein dynamics based tools to examine the significance of allosteric interactions and their influence on protein function.
ContributorsKazan, Ismail Can (Author) / Ozkan, Sefika Banu (Thesis advisor) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2023

An Investigation into the LLPS Effects of Radical Interactions on Intrinsically Disordered Proteins

Description

This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine…

This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine in various concentrations, and monitoring the output through UV-Vis spectroscopy. The reacted species was also analyzed via MALDI-TOF mass spectrometry. UV-Vis spectroscopic monitoring showed that in the presence of LAF-1 or tyrosine, the reaction between HRP and TMB occurred more quickly than the control, as well as in the highest concentration of LAF-1, the evolution of a peak at 482 nm. The analysis through MALDI-TOF spectrometry showed the development of a second peak likely due to the reaction between LAF-1 and TMB, as the Δ between the peaks is 229 Da and the size of the TMB species is 240 Da.
ContributorsDavis, Morgan (Author) / Ghirlanda, Giovanna (Thesis director) / Heyden, Matthias (Committee member) / Mazor, Yuval (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2022-12