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- All Subjects: Biophysics
- Creators: Ghirlanda, Giovanna
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
Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation
Description
Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.
ContributorsCope, Stephanie M (Author) / Vaiana, Sara M (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart M (Committee member) / Ozkan, Sefika B (Committee member) / Arizona State University (Publisher)
Created2013
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
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
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023
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 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
Description
Since understanding the nature of proteins, it has been a long held belief that protein sequence dictated structure which then determined function. As such, all proteins contained structure and those that did not must not serve a purpose. For the last 25 years, scientists have begun to understand that disordered proteins, lacking structure, did not lack function. Their unique ability to undergo liquid-liquid phase separation served a cellular purpose, most involving nucleic acids. As more is uncovered, these unique proteins are being used to build new systems. Phase separated disordered proteins were used to design a functional organelle using the enzyme horseradish peroxidase and its chromatic substrate ABTS. Upon doing so, it was discovered that disordered proteins are highly susceptible to chemical modification through radical reactions with tyrosine. The increased frequency of tyrosine in disordered proteins provides multiple sites of conjugation by the ABTS radical and other substrates. These modifications then alter the physical properties of the proteins. The phase separated system was also incorporated with shell proteins from bacterial microcompartments in an attempt to limit access to the droplets. Through expression with truncations of the disordered sequence, shell proteins were able to interact with the droplets. Despite the appearance of complete coatings, they were found to be permeable to their surroundings, though much more stable than uncoated droplets. Just as disordered proteins are considered outside the traditional structures, so too are many students entering higher education. Non-traditional students are becoming more prevalent in the undergraduate population, though they are woefully underrepresented in the natural sciences. The benefits these students bring to their programs is highlighted and the circumstances that drive them away from STEM is explored. Non-traditional students contribute to the diversity of the scientific population, though many pursue education in non-STEM fields. To support these students, focus is put on andragogy (the teaching of adults), rather than pedagogy (the teaching of children). Non-traditional students face isolation and discrimination that is not being addressed by higher education institutions, hindering their ability to succeed. Through infrastructure designed for adult learners, STEM fields can be diversified in non-traditional ways.
ContributorsCostantino, Michele (Author) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy (Committee member) / Matyushov, Dmitry (Committee member) / Arizona State University (Publisher)
Created2024
Description
All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play diverse roles in physiology. Among the 27 TRP channels found in humans and other animals, TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1) are the primary sensors of cold and hot temperatures, respectively. They underlie the molecular basis of somatic temperature sensation, but beyond this are also known to be involved in body temperature and weight regulation, inflammation, migraine, nociception, and some types of cancer. Because of their broad physiological roles, these channels are an attractive target for potential therapeutic interventions.
This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding.
The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation.
This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding.
The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation.
ContributorsHilton, Jacob Kenneth (Author) / Van Horn, Wade D (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2019
Description
Molecular docking serves as an important tool in modeling protein-ligand interactions. Most of the docking approaches treat the protein receptor as rigid and move the ligand in the binding pocket through an energy minimization, which is an incorrect approach as proteins are flexible and undergo conformational changes upon ligand binding. However, modeling receptor backbone flexibility in docking is challenging and computationally expensive due to the large conformational space that needs to be sampled.
A novel flexible docking approach called BP-Dock (Backbone Perturbation docking) was developed to overcome this challenge. BP-Dock integrates both backbone and side chain conformational changes of a protein through a multi-scale approach. In BP-Dock, the residues along a protein chain are perturbed mimicking the binding induced event, with a small Brownian kick, one at a time. The fluctuation response profile of the chain upon these perturbations is computed by Perturbation Response Scanning (PRS) to generate multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, this approach was applied to a large and diverse dataset of unbound structures as receptors. Furthermore, the protein-peptide docking of PICK1-PDZ proteins was investigated. This study elucidates the determinants of PICK1-PDZ binding that plays crucial roles in numerous neurodegenerative disorders. BP-Dock approach was also extended to the challenging problem of protein-glycan docking and applied to analyze the energetics of glycan recognition in Cyanovirin-N (CVN), a cyanobacterial lectin that inhibits HIV by binding to its highly glycosylated envelope protein gp120. This study provide the energetic contribution of the individual residues lining the binding pocket of CVN and explore the effect of structural flexibility in the hinge region of CVN on glycan binding, which are also verified experimentally. Overall, these successful applications of BP-Dock highlight the importance of modeling backbone flexibility in docking that can have important implications in defining the binding properties of protein-ligand interactions.
Finally, an induced fit docking approach called Adaptive BP-Dock is presented that allows both protein and ligand conformational sampling during the docking. Adaptive BP-Dock can provide a faster and efficient docking approach for the virtual screening of novel targets for rational drug design and aid our understanding of protein-ligand interactions.
A novel flexible docking approach called BP-Dock (Backbone Perturbation docking) was developed to overcome this challenge. BP-Dock integrates both backbone and side chain conformational changes of a protein through a multi-scale approach. In BP-Dock, the residues along a protein chain are perturbed mimicking the binding induced event, with a small Brownian kick, one at a time. The fluctuation response profile of the chain upon these perturbations is computed by Perturbation Response Scanning (PRS) to generate multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, this approach was applied to a large and diverse dataset of unbound structures as receptors. Furthermore, the protein-peptide docking of PICK1-PDZ proteins was investigated. This study elucidates the determinants of PICK1-PDZ binding that plays crucial roles in numerous neurodegenerative disorders. BP-Dock approach was also extended to the challenging problem of protein-glycan docking and applied to analyze the energetics of glycan recognition in Cyanovirin-N (CVN), a cyanobacterial lectin that inhibits HIV by binding to its highly glycosylated envelope protein gp120. This study provide the energetic contribution of the individual residues lining the binding pocket of CVN and explore the effect of structural flexibility in the hinge region of CVN on glycan binding, which are also verified experimentally. Overall, these successful applications of BP-Dock highlight the importance of modeling backbone flexibility in docking that can have important implications in defining the binding properties of protein-ligand interactions.
Finally, an induced fit docking approach called Adaptive BP-Dock is presented that allows both protein and ligand conformational sampling during the docking. Adaptive BP-Dock can provide a faster and efficient docking approach for the virtual screening of novel targets for rational drug design and aid our understanding of protein-ligand interactions.
ContributorsBolia, Ashini (Author) / Ozkan, Sefika Banu (Thesis advisor) / Ghirlanda, Giovanna (Thesis advisor) / Beckstein, Oliver (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2015
Description
This thesis explores a wide array of topics related to the protein folding problem, ranging from the folding mechanism, ab initio structure prediction and protein design, to the mechanism of protein functional evolution, using multi-scale approaches. To investigate the role of native topology on folding mechanism, the native topology is dissected into non-local and local contacts. The number of non-local contacts and non-local contact orders are both negatively correlated with folding rates, suggesting that the non-local contacts dominate the barrier-crossing process. However, local contact orders show positive correlation with folding rates, indicating the role of a diffusive search in the denatured basin. Additionally, the folding rate distribution of E. coli and Yeast proteomes are predicted from native topology. The distribution is fitted well by a diffusion-drift population model and also directly compared with experimentally measured half life. The results indicate that proteome folding kinetics is limited by protein half life. The crucial role of local contacts in protein folding is further explored by the simulations of WW domains using Zipping and Assembly Method. The correct formation of N-terminal β-turn turns out important for the folding of WW domains. A classification model based on contact probabilities of five critical local contacts is constructed to predict the foldability of WW domains with 81% accuracy. By introducing mutations to stabilize those critical local contacts, a new protein design approach is developed to re-design the unfoldable WW domains and make them foldable. After folding, proteins exhibit inherent conformational dynamics to be functional. Using molecular dynamics simulations in conjunction with Perturbation Response Scanning, it is demonstrated that the divergence of functions can occur through the modification of conformational dynamics within existing fold for β-lactmases and GFP-like proteins: i) the modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate, while the resurrected ancient lactamases indicate enhanced active-site flexibility, which likely allows for the binding and subsequent degradation of different antibiotic molecules; ii) the chromophore and attached peptides of photocoversion-competent GFP-like protein exhibits higher flexibility than the photocoversion-incompetent one, consistent with the evolution of photocoversion capacity.
ContributorsZou, Taisong (Author) / Ozkan, Sefika B (Thesis advisor) / Thorpe, Michael F (Committee member) / Woodbury, Neal W (Committee member) / Vaiana, Sara M (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014