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- All Subjects: Biophysics
- Creators: Chiu, Po-Lin
Description
The properties of materials depend heavily on the spatial distribution and connectivity of their constituent parts. This applies equally to materials such as diamond and glasses as it does to biomolecules that are the product of billions of years of evolution. In science, insight is often gained through simple models with characteristics that are the result of the few features that have purposely been retained. Common to all research within in this thesis is the use of network-based models to describe the properties of materials. This work begins with the description of a technique for decoupling boundary effects from intrinsic properties of nanomaterials that maps the atomic distribution of nanomaterials of diverse shape and size but common atomic geometry onto a universal curve. This is followed by an investigation of correlated density fluctuations in the large length scale limit in amorphous materials through the analysis of large continuous random network models. The difficulty of estimating this limit from finite models is overcome by the development of a technique that uses the variance in the number of atoms in finite subregions to perform the extrapolation to large length scales. The technique is applied to models of amorphous silicon and vitreous silica and compared with results from recent experiments. The latter part this work applies network-based models to biological systems. The first application models force-induced protein unfolding as crack propagation on a constraint network consisting of interactions such as hydrogen bonds that cross-link and stabilize a folded polypeptide chain. Unfolding pathways generated by the model are compared with molecular dynamics simulation and experiment for a diverse set of proteins, demonstrating that the model is able to capture not only native state behavior but also partially unfolded intermediates far from the native state. This study concludes with the extension of the latter model in the development of an efficient algorithm for predicting protein structure through the flexible fitting of atomic models to low-resolution cryo-electron microscopy data. By optimizing the fit to synthetic data through directed sampling and context-dependent constraint removal, predictions are made with accuracies within the expected variability of the native state.
ContributorsDe Graff, Adam (Author) / Thorpe, Michael F. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Matyushov, Dmitry (Committee member) / Ozkan, Sefika B. (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs.
Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
ContributorsNandi, Purbasha (Author) / Chiu, Po-Lin (Thesis advisor) / Mazor, Yuval (Committee member) / Hansen, Debra T (Committee member) / Arizona State University (Publisher)
Created2022
Description
Protein interactions with the environment are crucial for proper function, butinteraction mechanisms are not always understood. In G protein-coupled receptors
(GPCRs), cholesterol modulates the function in some, but not all, GPCRs. Coarse
grained molecular dynamics was used to determine a set of contact events for each
residue and fit to a biexponential to determine the time scale of the long contacts
observed in simulation. Several residues of interest were indicated in CCK1 R near
Y140, which is known to render CCK1 R insensitive to cholesterol when mutated to
alanine. A difference in the overall residence time between CCK1 R and its cholesterol
insensitive homologue CCK2 R was also observed, indicating the ability to predict
relative cholesterol binding for homologous proteins.
Occasionally large errors and poor fits to the data were observed, so several
improvements were made, including generalizing the model to include K exponential
components. The sets of residence times in the improved method were analyzed using
Bayesian nonparametrics, which allowed for error estimations and the classification of
contact events to the individual components. Ten residues in three GPCRs bound to
cholesterol in experimental structures had large tau. Slightly longer overall interaction
time for the cholesterol sensitive CB1 R over its insensitive homologue CB2 R was also
observed.
The interactions between the cystic fibrosis transmembrane conductance regulator
(CFTR) and GlyH-101, an open-channel blocker, were analyzed using molecular
dynamics. The results showed the bromine in GlyH-101 was in constant contact with
F337, which is just inside the extracellular gate. The simulations also showed an
insertion of GlyH-101 between TM1 and TM6 deeper than the starting binding pose.
Once inserted deeper between TMs 1 and 6, the number of persistent contacts also
increased. This proposed binding pose may help in future investigations of CFTR
and help determine an open-channel structure for the protein, which in turn may help
in the development of treatments for various medical conditions. Overall, the use
of molecular dynamics and state of the art analysis tools can be useful in the study
of membrane proteins and eventuallyin the development of treatments for ailments
stemming from their atypical function.
ContributorsSexton, Ricky (Author) / Beckstein, Oliver (Thesis advisor) / Presse, Steve (Committee member) / Ozkan, Sefika B. (Committee member) / Hariadi, Rizal (Committee member) / Arizona State University (Publisher)
Created2022
Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
ContributorsLuu, Dustin Dean (Author) / Van Horn, Wade D (Thesis advisor) / Redding, Kevin E (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic.
Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane.
Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are a large family of proteins involved in the cell signaling and regulation of many biological and pathological processes in the human body. To fully understand their functions, various approaches are needed. This work combines several techniques to advance the study of GPCRs with the overarching goal of pursuing X-ray crystallization using lipidic cubic phase (LCP). In meso, or LCP crystallization method involves imbedding the GPCR into a lipid membrane-mimetic material which spontaneously forms when monoacylglycerols (MAGs) are mixed at the correct hydration level and temperature. It provides a stable environment for GPCRs and has been established as the most common method to resolve structural details of GPCRs (Chapter 2). Yet, before crystallization, GPCRs need to be put through several rounds of optimization of the construct design, including truncation of N- and C- termini, fusing different soluble proteins, and mutating the receptor (Chapter 3). Other methods were also used to gain structural insights into GPCR interactions, such as coarse-grained molecular dynamic simulations, which showed the specific regions of interactions with cholesterol molecules imbedded in the membranes (Chapter 4). This study demonstrated β2-adrenergic receptor (β2AR), a GPCR, as a model of a cholesterol-sensitive receptor. Mutations were made to test the effect of removing specific residues of interest on cholesterol stabilization through the LCP-Tm assay, producing results that align with the simulation data. Finally, the goal of the last study is to provide a guide to identify which host lipids form stable LCP phases for different applications (Chapter 5). Small angle X-ray scattering is used to identify phases in hundreds of different precipitant conditions in the search of suitable host lipid for LCP studies. The results present a systematic overview of the compatibility of common MAGs by screening them against different precipitant solutions including varying salts and polyethylene glycol (PEG) concentrations, different PEG sizes, the presence of detergent or protein in the sample, and the addition of cholesterol. Together, these studies present a variety of methods to advance the structural studies of GPCRs using LCP
ContributorsAL-SAHOURI, ZINA (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021