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- All Subjects: Biophysics
- Creators: Beckstein, Oliver
- Creators: Levitus, Marcia
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
This dissertation describes the work on two projects which involves measuring molecular conductance and studying their properties on the nanoscale using various Scanning Tunneling Microscopy (STM) techniques. The first molecule studied was a porphyrin-fullerene moiety known as a molecular Dyad for photovoltaic applications. This project is further divided into two section, the first one involving the characterization of the Dyad monolayers and conductance measurement in the dark. The Dyads are designed to form charge separated states on illumination. The lifetime of the charged states have been measured efficiently but the single-molecule conductance through the molecules have yet to be characterized. The second part of the project describes the set-up of a novel sample stage which enables the study of molecular conductance under illumination. This part also describes the subsequent study of the molecule under illumination and the observation of a unique charge-separated state. It also contains the verification of the presence of this charge-separated using other characterization techniques like transient absorption spectroscopy. The second project described in the dissertation was studying and comparing the predicted rectifying nature of two molecules, identical in every way except for one stereocenter. This project describes the formation of monolayers of the molecule on gold and then studying and analyzing the current-voltage characteristics of the molecules and looking for rectification. Both the molecules proved to be rectifying, one more than the other as predicted by theoretical calculations.
ContributorsBhattacharyya, Shreya (Author) / Lindsay, Stuart (Thesis advisor) / Moore, Ana (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Single molecule DNA Sequencing technology has been a hot research topic in the recent decades because it holds the promise to sequence a human genome in a fast and affordable way, which will eventually make personalized medicine possible. Single molecule differentiation and DNA translocation control are the two main challenges in all single molecule DNA sequencing methods. In this thesis, I will first introduce DNA sequencing technology development and its application, and then explain the performance and limitation of prior art in detail. Following that, I will show a single molecule DNA base differentiation result obtained in recognition tunneling experiments. Furthermore, I will explain the assembly of a nanofluidic platform for single strand DNA translocation, which holds the promised to be integrated into a single molecule DNA sequencing instrument for DNA translocation control. Taken together, my dissertation research demonstrated the potential of using recognition tunneling techniques to serve as a general readout system for single molecule DNA sequencing application.
ContributorsLiu, Hao (Author) / Lindsay, Stuart M (Committee member) / Yan, Hao (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2013
Description
Transportation of material across a cell membrane is a vital process for maintaininghomeostasis. Na+/H+ antiporters, for instance, help maintain cell volume and regulate
intracellular sodium and proton concentrations. They are prime drug targets, since
dysfunction of these crucial proteins in humans is linked to heart and neurodegenerative
diseases. Due to their placement in a cell membrane, their study is particularly difficult
compared to globular proteins, which is likely the reason the transport mechanisms
for these proteins are not entirely known. This work focuses on the electrogenic
bacterial homologs Thermus thermophilus NapA (TtNapA) and Echerichia coli NhaA
(EcNhaA), each transporting one sodium from the interior of the cell for two protons
on outside of the cell. Even though X-ray crystal structures for both of these systems
have been resolved, their study through molecular dynamics (MD) simulations is
limited. The dynamic protonation and deprotonation of the binding site residues is
a fundamental process in the transport cycle, which currently cannot be explored
intuitively with standard MD methodologies. Apart from this limitation, simulation
performance is only a fraction of what is needed to understand the full transport
process, particularly when it comes to global conformational changes. This work
seeks to overcome these limitations through the development and application of a
multiscale thermodynamic and kinetic framework for constructing models capable of
predicting experimental observables, such as the dependence of transporter turnover
on membrane voltage. These models allow interpretation of the effects of individual
processes on the function as a whole. This procedure is demonstrated for TtNapA and
the connection between structure and function is shown by computing cycle turnover
across a range of non-equilibrium conditions.
ContributorsKenney, Ian Michael (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Sefika Banu (Committee member) / Heyden, Matthias (Committee member) / Vaiana, Sara (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
Transition metal ions such as Zn2+, Mn2+, Co2+, and Fe2+ play crucial roles in organisms from all kingdoms of life. The homeostasis of these ions is mainly regulated by a group of secondary transporters from the cation diffusion facilitator (CDF) family. The mammalian zinc transporters (ZnTs), a subfamily of CDF, have been an important target for study as they are associated with several diseases, such as diabetes, delayed growth and osteopenia, Alzheimer’s disease, and Parkinsonism. The bacterial homolog of ZnTs, YiiP, is the first CDF transporter with a determined structure and is used as a model for studying the structural and mechanistic properties of CDF transporters. On the other hand, Molecular dynamics simulation has emerged as a valuable computational tool for exploring the physical basis of biological macromolecules' structure and function with atomic precision at femtosecond resolution. This work aims to elucidate the roles of the three Zn$2+ binding sites found on each YiiP protomer and the role of protons in the transport process of CDFs, which remain under debate despite previous thermodynamic and structural studies on YiiP. Cryo-EM, microscale thermophoresis (MST) and molecular dynamics (MD) simulations were used to address these questions. With a Zn2+ model that accurately reproduces experimental structures of the binding clusters, the dynamical influence of zinc binding on the transporter was accessed through MD simulations, which was consistent with the new cryo-EM structures. Zinc binding affinities obtained through MST were used to infer the stoichiometry of Zn2+/H+ antiport in combination with a microscopic thermodynamic model and constant pH simulations. The most likely microstates of H$^+$ and Zn2+ binding indicated a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. A model describing the entire transport cycle of YiiP was finally built on these findings, providing insight into the structural and mechanistic properties of CDF transporters.
ContributorsFan, Shujie (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Banu (Committee member) / Heyden, Matthias (Committee member) / Van Horn, Wade (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
Secondary active transporters play significant roles in maintaining living cells' homeostasis by utilizing the electrochemical gradient in driving ions or protons as the source of free energy to transport substrate through biological membranes.A broadly recognized molecular framework, the alternating access model, describes the transport mechanism as the transporter undergoes conformational changes between different conformations and alternatingly exposes its binding site to intracellular and extracellular sides and, thus, exchanges ion and substrate in a cyclical manner.
Recent progress in structural biology brought the first-ever structural insights into the mammalian Cation-Proton Antiporters (CPA) family of proteins.
However, the dynamic atomic-level information about the interactions between the newly discovered structures and the bound ion or the corresponding substrate remains unknown.
With Molecular Dynamics (MD), multiple spontaneous ion binding events were observed in the equilibrium simulations, revealing the binding site topology of Horse Sodium-Proton Exchanger 9 (NHE9) and Bison Sodium-Proton Antiporter 2 (NHA2) in their preferred protonation state.
Further investigation into more CPA homologs compared various aspects, including sequence identity, binding site topology, and energetic properties, and obtained general insights into the similarities shared by the binding process of CPA members.
The putative binding site and other conserved residues in their actively ion-bound poses were identified for each model, and their similarities were compared.
The energetic properties accessed by the three-dimensional free energy profile, initially found to be binding unfavorable for the experimental structures, were recalculated based on the simulation data. The updated results show consistency with the correct binding affinity as indicated by the experimental methods.
This work provided a general picture of the structures and the ion-protein interaction of CPA proteins and serves as comprehensive guidance for any related future structural and computational work.
ContributorsZhang, Chenou (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Banu (Committee member) / Ros, Robert (Committee member) / Singharoy, Abhishek (Committee member) / Arizona State University (Publisher)
Created2023
Description
Proteins, the machinery of life, perform a vast array of essential biochemical functions, evolving over time to acquire diverse roles within biological systems. This evolution, primarily driven by mutations within protein sequences, can profoundly impact protein function, potentially leading to various diseases. This thesis aims to dissect the intricate mechanisms through which genetic mutations influence protein functionality, focusing on the dynamic alterations induced by single and combined mutations. Employing a suite of computational tools, including molecular dynamics (MD) simulations and proven analysis metrics like the Dynamic Flexibility Index (DFI) and Dynamic Coupling Index (DCI), I analyze protein dynamics to uncover the common dynamic effects associated with disease causation and compensatory mechanisms. This analysis extends to exploring the concept of epistasis through the lens of protein dynamics, showing how combinations of mutations interact within the protein's 3D structure to either exacerbate or mitigate the functional impacts of individual mutations. The use of EpiScore, a computational tool designed to quantify the epistatic effects of mutations, provides insight on the combined dynamic effects two mutations might have. This is particularly evident in the analysis of rare alleles within human populations, where certain allele combinations, despite their individual rarity, frequently co-occur, suggesting a mechanism of dynamic compensation. This phenomenon is further investigated in the context of the SARS-CoV-2 spike protein, providing insights into viral evolution and the adaptive significance of specific mutations. Additionally, I delve into the role of Intrinsically Disordered Regions (IDRs) in protein function and mutation compensation, highlighting the need for sophisticated dynamics analysis tools to capture the full spectrum of mutation effects. By integrating these analyses, this thesis unveils a complex picture of how proteins' dynamic properties, shaped by mutations, underpin their functional evolution and disease outcomes.
ContributorsOse, Nicholas James (Author) / Ozkan, Sefika Banu (Thesis advisor) / Hariadi, Rizal (Committee member) / Beckstein, Oliver (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2024