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- All Subjects: Biophysics
- Creators: Yan, Hao
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
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 ⁰C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic γ-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability.
ContributorsFlory, Justin David (Author) / Fromme, Petra (Thesis advisor) / Yan, Hao (Committee member) / Buttry, Daniel (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
ABSTRACT
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
ContributorsWang, Dingjie (Author) / Spence, John CH (Thesis advisor) / Weierstall, Uwe (Committee member) / Schmidt, Kevin (Committee member) / Fromme, Petra (Committee member) / Ozkan, Banu (Committee member) / Arizona State University (Publisher)
Created2014
Description
Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These new methods include serial femtosecond crystallography, single particle imaging, solution scattering, and time resolved techniques.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.
ContributorsJames, Daniel (Author) / Spence, John (Thesis advisor) / Weierstall, Uwe (Committee member) / Kirian, Richard (Committee member) / Schmidt, Kevin (Committee member) / Arizona State University (Publisher)
Created2015
Description
The superior brightness and ultra short pulse duration of X-ray free electron laser
(XFEL) allows it to outrun radiation damage in coherent diffractive imaging since elastic scattering terminates before photoelectron cascades commences. This “diffract-before-destroy” feature of XFEL opened up new opportunities for biological macromolecule imaging and structure studies by breaking the limit to spatial resolution imposed by the maximum dose that is allowed before radiation damage. However, data collection in serial femto-second crystallography (SFX) using XFEL is affected by a bunch of stochastic factors, which pose great challenges to the data analysis in SFX. These stochastic factors include crystal size, shape, random orientation, X-ray photon flux, position and energy spectrum. Monte-Carlo integration proves effective and successful in extracting the structure factors by merging all diffraction patterns given that the data set is sufficiently large to average out all stochastic factors. However, this approach typically requires hundreds of thousands of patterns collected from experiments. This dissertation explores both experimental and algorithmic methods to eliminate or reduce the effect of stochastic factors in data acquisition and analysis. Coherent convergent X-ray beam diffraction (CCB) is discussed for possibilities of obtaining single-shot angular-integrated rocking curves. It is also shown the interference between Bragg disks helps ab-initio phasing. Two-color diffraction scheme is proposed for time-resolved studies and general data collection strategies are discussed based on error metrics. A new auto-indexing algorithm for sparse patterns is developed and demonstrated for both simulated and experimental data. Statistics show that indexing rate is increased by 3 times for I3C data set collected from beam time LJ69 at Linac coherent light source (LCLS). Finally, dynamical inversion from electron diffraction is explored as an alternative approach for structure determination.
(XFEL) allows it to outrun radiation damage in coherent diffractive imaging since elastic scattering terminates before photoelectron cascades commences. This “diffract-before-destroy” feature of XFEL opened up new opportunities for biological macromolecule imaging and structure studies by breaking the limit to spatial resolution imposed by the maximum dose that is allowed before radiation damage. However, data collection in serial femto-second crystallography (SFX) using XFEL is affected by a bunch of stochastic factors, which pose great challenges to the data analysis in SFX. These stochastic factors include crystal size, shape, random orientation, X-ray photon flux, position and energy spectrum. Monte-Carlo integration proves effective and successful in extracting the structure factors by merging all diffraction patterns given that the data set is sufficiently large to average out all stochastic factors. However, this approach typically requires hundreds of thousands of patterns collected from experiments. This dissertation explores both experimental and algorithmic methods to eliminate or reduce the effect of stochastic factors in data acquisition and analysis. Coherent convergent X-ray beam diffraction (CCB) is discussed for possibilities of obtaining single-shot angular-integrated rocking curves. It is also shown the interference between Bragg disks helps ab-initio phasing. Two-color diffraction scheme is proposed for time-resolved studies and general data collection strategies are discussed based on error metrics. A new auto-indexing algorithm for sparse patterns is developed and demonstrated for both simulated and experimental data. Statistics show that indexing rate is increased by 3 times for I3C data set collected from beam time LJ69 at Linac coherent light source (LCLS). Finally, dynamical inversion from electron diffraction is explored as an alternative approach for structure determination.
ContributorsLi, Chufeng (Author) / Spence, John CH (Thesis advisor) / Spence, John (Committee member) / Kirian, Richard (Committee member) / Weierstall, Uwe (Committee member) / Schmidt, Kevin (Committee member) / Arizona State University (Publisher)
Created2016
Description
Phase problem has been long-standing in x-ray diffractive imaging. It is originated from the fact that only the amplitude of the scattered wave can be recorded by the detector, losing the phase information. The measurement of amplitude alone is insufficient to solve the structure. Therefore, phase retrieval is essential to structure determination with X-ray diffractive imaging. So far, many experimental as well as algorithmic approaches have been developed to address the phase problem. The experimental phasing methods, such as MAD, SAD etc, exploit the phase relation in vector space. They usually demand a lot of efforts to prepare the samples and require much more data. On the other hand, iterative phasing algorithms make use of the prior knowledge and various constraints in real and reciprocal space. In this thesis, new approaches to the problem of direct digital phasing of X-ray diffraction patterns from two-dimensional organic crystals were presented. The phase problem for Bragg diffraction from two-dimensional (2D) crystalline monolayer in transmission may be solved by imposing a compact support that sets the density to zero outside the monolayer. By iterating between the measured stucture factor magnitudes along reciprocal space rods (starting with random phases) and a density of the correct sign, the complex scattered amplitudes may be found (J. Struct Biol 144, 209 (2003)). However this one-dimensional support function fails to link the rod phases correctly unless a low-resolution real-space map is also available. Minimum prior information required for successful three-dimensional (3D) structure retrieval from a 2D crystal XFEL diffraction dataset were investigated, when using the HIO algorithm. This method provides an alternative way to phase 2D crystal dataset, with less dependence on the high quality model used in the molecular replacement method.
ContributorsZhao, Yun (Author) / Spence, John C.H. (Thesis advisor) / Schmidt, Kevin (Committee member) / Weierstall, Uwe (Committee member) / Kirian, Richard (Committee member) / Zatsepin, Nadia (Committee member) / Arizona State University (Publisher)
Created2016
Description
The structure-function relation in Biology suggests that every biological molecule has evolved its structure to carry out a specific function. However, for many of these processes (such as those with catalytic activity) the structure of the biomolecule changes during the course of a reaction. Understanding the structure-function relation thus becomes a question of understanding biomolecular dynamics that span a variety of timescales (from electronic rearrangements in the femtoseconds to side-chain alteration in the microseconds and more). This dissertation deals with the study of biomolecular dynamics in the ultrafast timescales (fs-ns) using electron and X-ray probes in both time and frequency domains.
It starts with establishing the limitations of traditional electron diffraction coupled with molecular replacement to study biomolecular structure and proceeds to suggest a pulsed electron source Hollow-Cone Transmission Electron Microscope as an alternative scheme to pursue ultrafast biomolecular imaging. In frequency domain, the use of Electron Energy Loss Spectroscopy as a tool to access ultrafast nuclear dynamics in the steady state, is detailed with the new monochromated NiON UltraSTEM and examples demonstrating this instrument’s capability are provided.
Ultrafast X-ray spectroscopy as a tool to elucidate biomolecular dynamics is presented in studying X-ray as a probe, with the study of the photolysis of Methylcobalamin using time-resolved laser pump – X-ray probe absorption spectroscopy. The analysis in comparison to prior literature as well as DFT based XAS simulations offer good agreement and understanding to the steady state spectra but are so far inadequate in explaining the time-resolved data. However, the trends in the absorption simulations for the transient intermediates show a strong anisotropic dependence on the axial ligation, which would define the direction for future studies on this material to achieve a solution.
It starts with establishing the limitations of traditional electron diffraction coupled with molecular replacement to study biomolecular structure and proceeds to suggest a pulsed electron source Hollow-Cone Transmission Electron Microscope as an alternative scheme to pursue ultrafast biomolecular imaging. In frequency domain, the use of Electron Energy Loss Spectroscopy as a tool to access ultrafast nuclear dynamics in the steady state, is detailed with the new monochromated NiON UltraSTEM and examples demonstrating this instrument’s capability are provided.
Ultrafast X-ray spectroscopy as a tool to elucidate biomolecular dynamics is presented in studying X-ray as a probe, with the study of the photolysis of Methylcobalamin using time-resolved laser pump – X-ray probe absorption spectroscopy. The analysis in comparison to prior literature as well as DFT based XAS simulations offer good agreement and understanding to the steady state spectra but are so far inadequate in explaining the time-resolved data. However, the trends in the absorption simulations for the transient intermediates show a strong anisotropic dependence on the axial ligation, which would define the direction for future studies on this material to achieve a solution.
ContributorsSubramanian, Ganesh (Author) / Spence, John (Thesis advisor) / Rez, Peter (Committee member) / Alford, Terry (Committee member) / Weierstall, Uwe (Committee member) / Kirian, Richard (Committee member) / Arizona State University (Publisher)
Created2016
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