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Open Access Publications from the University of California

Scholarly Works (51 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2007)
Marine CaCO₃ skeletons have tailored architectures created by nature, which give them structural support and other functions. For example, seashells have dense lamellar structures, while coral, cuttlebone and sea urchin spines have interconnected porous structures. In our experiments, seashells, coral and cuttlebone were hydrothermally converted to hydroxyapatite (HAP), and sea urchin spines were converted to Mg-substituted tricalcium phosphate [beta]-TCMP, while maintaining their original structures. Partially converted shell samples have mechanical strength, which is close to that of compact human bone. After implantation of converted shell and spine samples in rat femoral defects for 6 weeks, there was newly formed bone growth up to and around the implants. Some new bone was found to migrate through the pores of converted spine samples and grow inward. These results show good bioactivity and osteoconductivity of the implants, indicating the converted shell and spine samples can be used as bone defect fillers. Calcium phosphate powders were prepared through different synthesis methods. Micro- size HAP rods were synthesized by hydrothermal method through a nucleation-growth mechanism. On the other hand, HAP particles, which have good crystallinity, were prepared by wet precipitation with further hydrothermal treatment. [beta]-TCP or [beta]-TCMP powders were prepared by a two-step process: wet precipitation of apatitic tricalcium phosphate ('precursor') and calcination of the precursor at 800⁰C for 3 hours. [beta]-TCMP or [beta]-TCP powders were also prepared by solid-state reactions from CaHPO₄ and CaCO₃ with/without MgO. Biphasic calcium phosphate, which is mixture of HAP and [beta]-TCP, can be prepared though mechanical mixing of HAP and [beta]-TCP powders synthesized as above. Dense [beta]-TCP and [beta]- TCMP ceramics can be produced by pressing green bodies at 100MPa and further sintering above 1100⁰C for 2 hours. [beta]-TCMP ceramics ̃99.4% relative dense were prepared by this method. Dense [beta]-TCP ceramics have average strength up to 540MPa. Macroporous [beta]-TCMP ceramics were produced with sucrose as the porogen following a two- step pressing method. Porous [beta]-TCMP ceramics were also prepared by replication of polyurethane sponge. [beta]-TCMP ceramics with porous structures in the center surrounded by dense structures were created. The outside dense structures give the scaffold mechanical strength, while the central porous structures enable cells migration and vascular infiltration, and finally in-growth of new bone into the scaffold
    Cover page: Preparation and characterization of calcium phosphate ceramics and Composites as bone substitutes
    • Thesis
    • Peer Reviewed
    UC Riverside Electronic Theses and Dissertations (2014)

    Mass spectrometry (MS) is best known for measuring molecular weight and probing the chemical structure. Radical directed dissociation (RDD) is a tandem MS fragmentation method for hydrogen deficient radical precursors, which provides complementary structure information compared to other tandem MS methods.

    Fragmentation pathways of peptide radical isomers in collision induced dissociation are radical site dependent. The purpose of my first project is to use RDD fragmentation patterns from peptide radical isomers as probes of peptides gas phase structure and conformation flexibility. Radicals are site-specifically generated by photodissociation of carbon-iodine bonds at ortho-, meta-, or para- position of a benzoyl ring labeled at peptide amine group. Radicals at these sites have almost identical bond dissociation energies and thus the only difference comes from the steric effect. We utilized the steric effect and isotope deuterium labeling to study peptide gas phase structure and its influence on intramolecular radical migration.

    Evaluating protein structure in the gas phase is useful for understanding the intrinsic forces which influence protein folding and for determining the feasibility of probing condensed phase structure with gas phase interrogation. KIX is a three-helix bundle protein that has been reported previously to preserve the condensed phase structure in the gas phase. In another project, structure dependent RDD is used to examine the gas phase structure of KIX by establishing residue specific distance constraints which can be used to assess candidate structures obtained from molecular dynamics simulations. The data obtained by RDD is consistent with KIX structures that largely retain condensed phase structure as determined previously by NMR.

    A new method is developed to probe peptide structure in vacuo utilizing di-radical recombination reaction. Commonly, the pairing of electrons which occurs when two radicals interact is a radical exothermic process without an activation barrier. In the case of a separated peptide ion containing two radicals in the gas phase, such radical coupling reaction is an intramolecular cyclization process and generates a cyclic peptide. Therefore, examining the sites where the di-radical recombination occurs can provide local contact information. Herein, two radical precursors are site specifically introduced to polyproline at the N and C terminal residue, respectively. Internal fragments from cyclized polyproline peptides and ion-molecule reaction data indicate that polyprolines no longer adopts a helical structure as in the solution which would prevent diradical interaction.

    Intermolecular homolytic aromatic substitution (HAS) reaction between benzoate phenyl radical and aromatic side chains in large peptide is observed and investigated. Compared to those radical precursors we previously used, 2-iodo benzoic acid has a much higher propensity to add to the aromatic rings in peptides. HAS is in competitive with radical migration reaction and the product branching ratio depends on specific complex structure. The reaction mechanisms are verified by labeling at various key positions for tryptophan and tyrosine. Novel side chain losses from aromatic residues (-116W, -93Y, -77F) are rationalized by HAS. Phenylalanine is a chemically inert residue. Such unique side chain loss (i.e., -77F) allows identification of phenylalanine residue in peptides. Study of phenyl radical damage to oligopeptides in the gas phase by radical substitution reaction is achieved for the first time.

    New methods are developed to dissociate oligosaccharides based on RDD. Radical saccharides can be generated by either intermolecular radical migrations or covalent labeling with radical precursors. Compared to CID of even electron saccharides, RDD provides rich fragmentations including cross-ring cleavages. Positional isomers of oligosaccharides (e.g., lacto-N-fucopentaose isomers and lacto-N-difucohexaose isomers) are able to be distinguished by RDD owing to observed signature fragments.

      Cover page: Structural Characterization of Proteins, Peptides and Oligosaccharides Based on Radical Chemistry and Mass Spectrometry
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2019)
      Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.
        Cover page: Synthesis of New S-S and C-C Bonds by Photoinitiated Radical Recombination Reactions in the Gas Phase.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2015)
        Geranyl-CoA carboxylase (GCC) is essential for the growth of Pseudomonas organisms with geranic acid as the sole carbon source. GCC has the same domain organization and shares strong sequence conservation with the related biotin-dependent carboxylases 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC). Here we report the crystal structure of the 750-kDa α6β6 holoenzyme of GCC, which is similar to MCC but strikingly different from PCC. The structures provide evidence in support of two distinct lineages of biotin-dependent acyl-CoA carboxylases, one carboxylating the α carbon of a saturated organic acid and the other carboxylating the γ carbon of an α-β unsaturated acid. Structural differences in the active site region of GCC and MCC explain their distinct substrate preferences. Especially, a glycine residue in GCC is replaced by phenylalanine in MCC, which blocks access by the larger geranyl-CoA substrate. Mutation of this residue in the two enzymes can change their substrate preferences.
          Cover page: Structure and substrate selectivity of the 750-kDa α6β6 holoenzyme of geranyl-CoA carboxylase
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2014)
          In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.
            • Article
            • Peer Reviewed
            LBL Publications (2016)
            Cholesteryl ester transfer protein (CETP) mediates cholesteryl ester (CE) transfer from the atheroprotective high density lipoprotein (HDL) cholesterol to the atherogenic low density lipoprotein cholesterol. In the past decade, this property has driven the development of CETP inhibitors, which have been evaluated in large scale clinical trials for treating cardiovascular diseases. Despite the pharmacological interest, little is known about the fundamental mechanism of CETP in CE transfer. Recent electron microscopy (EM) experiments have suggested a tunnel mechanism, and molecular dynamics simulations have shown that the flexible N-terminal distal end of CETP penetrates into the HDL surface and takes up a CE molecule through an open pore. However, it is not known whether a CE molecule can completely transfer through an entire CETP molecule. Here, we used all-atom molecular dynamics simulations to evaluate this possibility. The results showed that a hydrophobic tunnel inside CETP is sufficient to allow a CE molecule to completely transfer through the entire CETP within a predicted transfer time and at a rate comparable with those obtained through physiological measurements. Analyses of the detailed interactions revealed several residues that might be critical for CETP function, which may provide important clues for the effective development of CETP inhibitors and treatment of cardiovascular diseases.
              Cover page: Insights into the Tunnel Mechanism of Cholesteryl Ester Transfer Protein through All-atom Molecular Dynamics Simulations*
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2019)
              Pre-exposure prophylaxis (PrEP) with topically or systemically administered antiretroviral agents can prevent acquisition of human immunodeficiency virus type 1 (HIV-1) infection. However, in clinical trials using tenofovir-containing agents, HIV-1 acquisition is reduced but not eliminated. Incomplete adherence remains the major contributor to failure. Sustained release or long-acting antiretroviral agents may provide better HIV-1 protection by reducing the clinical impact of incomplete adherence. To reduce dosing frequency, we synthesized a novel tenofovir prodrug, octadecyloxyethyl benzyl tenofovir (ODE-Bn-TFV), that is designed to release TFV slowly in tissues, and showed potent anti-HIV activity in vitro (EC50 = 1.7 nM). In cells exposed to 14C labeled TFV, ODE-Bn-TFV or the quickly activated monoester ODE-TFV, rapid cellular uptake for both lipophilic analogs was noted, achieving 50-fold higher levels than unmodified TFV after 48 h. Following exposure to ODE-[8-14C]TFV, the intracellular diphosphate levels were approximately four-fold higher than with ODE-Bn-TFV. However, intracellular TFVpp drug levels fell rapidly yielding a half-life of about two days. TFVpp levels in ODE-Bn-TFV treated cells decreased much more slowly and reached half-maximal levels in about seven days. These results suggest early accumulation of ODE-Bn-TFV followed by sustained intracellular release following cleavage of the ester bonds linking the ODE and benzyl moieties to the active molecular precursor, thereby potentially allowing for less frequent administration than with more rapidly activated forms of tenofovir.
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2016)
                Viruses sense environmental cues such as pH to engage in membrane interactions for cell entry during infection, but how nonenveloped viruses sense pH is largely undefined. Here, we report both high- and low-pH structures of bluetongue virus (BTV), which enters cells via a two-stage endosomal process. The receptor-binding protein VP2 possesses a zinc finger that may function to maintain VP2 in a metastable state and a conserved His866, which senses early-endosomal pH. The membrane-penetration protein VP5 has three domains: dagger, unfurling and anchoring. Notably, the β-meander motif of the anchoring domain contains a histidine cluster that can sense late-endosomal pH and also possesses four putative membrane-interaction elements. Exposing BTV to low pH detaches VP2 and dramatically refolds the dagger and unfurling domains of VP5. Our biochemical and structure-guided-mutagenesis studies support these coordinated pH-sensing mechanisms.
                  Cover page: Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2006)
                  We present a model-based parallel algorithm for origin and orientation refinement for 3D reconstruction in cryoTEM. The algorithm is based upon the Projection Theorem of the Fourier Transform. Rather than projecting the current 3D model and searching for the best match between an experimental view and the calculated projections, the algorithm computes the Discrete Fourier Transform (DFT) of each projection and searches for the central section ("cut") of the 3D DFT that best matches the DFT of the projection. Factors that affect the efficiency of a parallel program are first reviewed and then the performance and limitations of the proposed algorithm are discussed. The parallel program that implements this algorithm, called PO(2)R, has been used for the refinement of several virus structures, including those of the 500 Angstroms diameter dengue virus (to 9.5 Angstroms resolution), the 850 Angstroms mammalian reovirus (to better than 7A), and the 1800 Angstroms paramecium bursaria chlorella virus (to 15 Angstroms).
                    Cover page: A model-based parallel origin and orientation refinement algorithm for cryoTEM and its application to the study of virus structures
                    • Article
                    • Peer Reviewed
                    UC Riverside Previously Published Works (2019)
                    Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.
                      Cover page: Synthesis of new SS and CC bonds by photoinitiated radical recombination reactions in the gas phase
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