Advanced Imaging Techniques for Point-Measurement Analysis of Pharmaceutical Materials

Drugs are an essential element protecting human lives from many diseases such as cancer, diabetes, and cardiovascular disorders. One of the highlights in drug development in recent years is the establishment of rational drug design: a collection of various multi-disciplinary approaches that at the core, focus on designing molecules with specific properties for identified targets and biomolecules with known functional roles and structural information. The candidate molecules will then go through a series of examinations to characterize their physiochemical properties, and an iterative process is used to improve the design of the drug to achieve desirable attributes. The time consuming and highly expensive nature of drug development constantly calls for new analytical techniques that have increasingly higher throughput, faster analysis speed, richer chemical and structural information, and lower risk and cost. Conventional analytical methods for pharmaceutical materials, such as X-ray diffraction analysis and Raman spectroscopy, often suffer from prolonged measurement time. In many cases, the identification of regions of interest within the sample is non-trivial in itself. Nonlinear optical imaging techniques, including second harmonic generation (SHG) microscopy and two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy were developed as fast, real-time, and non-destructive methods for selective identification and characterization of crystalline materials present in pharmaceutical samples. These techniques were integrated with synchrotron X-ray diffraction analysis and Raman spectroscopy to significantly reduce the overall measurement time of these structure characterization techniques. In the meanwhile, with the now increased speed of measurement, the amount of experimental data acquired per unit time has also drastically increased. The rate at which data are analyzed, digested, and interpreted is becoming the bottleneck in data-driving decision-making. Novel electronics that only collect data at the most information-rich time points were employed to significantly increase the signal-to-noise ratio (SNR) during data acquisition, reducing the total amount of data needed for material characterization. Advanced sampling algorithms to reduce the total amount of measurements required for perfect data space reconstruction, automated programs for data acquisition and analysis, and efficient data analysis algorithms based on machine learning were developed for accelerated data processing for nonlinear optical imaging analysis, Raman spectra processing, and X-ray diffraction indexing

Direct Phasing of Finite Crystals Illuminated with a Free-Electron Laser

abstract: It has been suggested that the extended intensity profiles surrounding Bragg reflections that arise when a series of finite crystals of varying size and shape are illuminated by the intense, coherent illumination of an x-ray free-electron laser may enable the crystal’s unit-cell electron density to be obtained ab initio via well-established iterative phasing algorithms. Such a technique could have a significant impact on the field of biological structure determination since it avoids the need for a priori information from similar known structures, multiple measurements near resonant atomic absorption energies, isomorphic derivative crystals, or atomic-resolution data. Here, we demonstrate this phasing technique on diffraction patterns recorded from artificial two-dimensional microcrystals using the seeded soft x-ray free-electron laser FERMI. We show that the technique is effective when the illuminating wavefront has nonuniform phase and amplitude, and when the diffraction intensities cannot be measured uniformly throughout reciprocal space because of a limited signal-to-noise ratio

Methods and Instrumentation of Sample Injection for XFEL Experiments

abstract: 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.Dissertation/ThesisDoctoral Dissertation Physics 201

Structural and biophysical analysis of the proteasomal deubiquitinase, UCH37

Ubiquitin carboxyl-terminal hydrolase 37, or UCH37, is a deubiquitinating enzyme associated with the 26S proteasome, the primary protein degradation machinery in eukaryotic cells. UCH37 is responsible for the disassembly of polymeric ubiquitin chains, or polyubiquitin, which have been ligated onto proteins in order to target them for degradation. The 26S utilizes two associated deubiquitinating enzymes, UCH37 and USP14, and one intrinsic, Rpn11, to remove polyubiquitin chains from substrate proteins as they are unfolded and translocated into the proteolytic core of the proteasome, where proteins are cleaved into small peptides and then released for recycling by the cell. UCH37 associates with the proteasome via binding of its C-terminal KEKE motif to the C-terminus of Rpn13, a proteasomal ubiquitin receptor which ensnares polyubiquitinated prey for degradation. UCH37 is known to be catalytically activated upon binding to Rpn13, allowing cleavage of Lys48-linked polyubiquitin chains from their distal end, an exo-specific deubiquitination. However, free UCH37 cleaves polyubiquitin poorly and is believed to be autoinhibited by its C-terminal UCHL5-like domain, or ULD, which may also be responsible for its oligomerization in solution. This work examines the structural, biophysical, and catalytic characteristics of UCH37 in order to elucidate its mechanism of activation by Rpn13, assess its biophysical assembly with Rpn13 within the greater proteasomal context, and ascertain its mechanism of exo-specificity despite the proteasome\u27s processing of a variety of polyubiquitinated substrates.^ To this end, a 1.7 Å resolution x-ray crystal structure was solved of the catalytic domain of a UCH37 homolog from Trichinella spiralisin complex with ubiquitin vinyl methyl ester (UbVME), a suicide inhibitor substrate. Our structure, in combination with another solved of a longer construct of TsUCH37 in complex with UbVME, provided structural insights into the ability of UCH37 to process polyubiquitin, namely that its C-terminal UCHL5-like domain (ULD) is responsible for its exo-specific activity due to a network of interactions with ubiquitin\u27s Lys48.^ Through biophysical and kinetic characterization, we have affirmed the poor activity of UCH37 alone, but do not ascribe it to autoinhibition because it does not oligomerize as previously thought, rather we find that it sediments in a monomer-dimer equilibrium in analytical ultracentrifugation experiments. We have characterized its binding and activation by Rpn13, finding that UCH37 binds to Rpn13 with a 22 nM dissociation constant and that mutations to UCH37\u27s ULD render it unable to be activated by Rpn13. Interestingly, we have found that while Rpn13 activates UCH37 for ubiquitin-AMC cleavage, a monoubiquitin fluorogenic substrate, it appears to slow the enzyme\u27s processing of Lys48-linked polyubiquitin chains in our assays.^ Altogether, we have confirmed that UCH37 exists primarily as a monomer which binds tightly to its proteasomal subunit, Rpn13, and can exo-specifically cleave Lys48-linked polyubiquitin chains. However, UCH37 may not be activated as was previously thought, by Rpn13 alone, and likely requires full association with the 26S proteasome

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

UNDERSTANDING AND EVALUATING CRYSTAL POLYMORPHISM BY SECOND HARMONIC GENERATION MICROSCOPY

The crystalline form of a solid can profoundly affect its physical and chemical properties, with both potentially stable and metastable crystal polymorphs are accessible during crystal formation. Conventional methods limit the detection of rare nucleation and rapid phase transitioning events due to their lack of selectivity and sensitivity. Inkjet printing of a solution confines the nucleation event in a few micrometer volumes within the droplet, and furthermore rapid desolvation favors the kinetic factor to trap the rare metastable polymorphs. Second harmonic generation microscopy (SHG) possesses enough sensitivity to detect sub-micrometer size chiral crystals selectively and has the potential for use in crystal nucleation studies

Rapid sample delivery for megahertz serial crystallography at X-ray FELs

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments

Rapid sample delivery for megahertz serial crystallography at X-ray FELs

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.Unión Europea 7PM / 2007-2013Consejo de Investigación de Australia DP170100131Ministerio de Economía, Industria y Competitividad DPI2016-78887-C3-1-RNational Science Foundation "BioXFEL" (1231306

Serial Electron Diffraction Data Processing With diffractem and CrystFEL

Serial electron diffraction (SerialED) is an emerging technique, which applies the snapshot data-collection mode of serial X-ray crystallography to three-dimensional electron diffraction (3D Electron Diffraction), forgoing the conventional rotation method. Similarly to serial X-ray crystallography, this approach leads to almost complete absence of radiation damage effects even for the most sensitive samples, and allows for a high level of automation. However, SerialED also necessitates new techniques of data processing, which combine existing pipelines for rotation electron diffraction and serial X-ray crystallography with some more particular solutions for challenges arising in SerialED specifically. Here, we introduce our analysis pipeline for SerialED data, and its implementation using the CrystFEL and diffractem program packages. Detailed examples are provided in extensive supplementary code

Polarization-dependent nonlinear optical microscopy methods for the analysis of crystals and biological tissues

The ability to solve a high-resolution protein structure is largely dependent on the successful generation and identification of protein crystals prior to X-ray diffraction (XRD). For novel protein targets, high-throughput crystallography often involves generation of multiple targets and thousands of crystallization trials per target to generate diffraction-quality crystals. Second harmonic generation (SHG) imaging has been developed as a fast, non-destructive and sensitive method for the selective identification of protein crystals, even in highly scattering environments. Polarization-dependent SHG microscopy methods were developed to assess the presence of multidomain crystals to provide a handle on crystal quality. In addition, polarization-dependent two-photon excited fluorescence (TPEF) microscopy was developed as a complementary method to SHG, providing selectivity based on the presence of protein and crystalline order, thereby reducing the potential for false negatives and positives that can arise with SHG and conventional TPEF imaging. Novel instrumentation, data acquisition methods, and data analysis techniques were developed for quantitative polarization-modulated SHG microscopy at imaging speeds up to video rate, offering significantly greater signal to noise ratios compared to polarization modulation through the manual rotation of wave plates. Quantitative polarization-dependent SHG imaging was extended to the analysis of collagen structures in biological tissues, where local-frame second order susceptibility tensors were solved for every pixel within an image of collagenous tissue and combined with ab initio modeling to assess internal ordering of collagen fibers in different tissue types