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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

Nature self-assembles protein structures for various functions, including: storage, protection, and fortification. These self-assemblies range from filaments to full three-dimensional crystals and are pervasive across the tree of life. They include the granules present in immune system cells, the packing of hormones in the pancreas, the storage of proteins in plants, carboxysomes, viruses, and cell-grown crystals in microbes. To accomplish this, a better understanding of how certain organisms are able to naturally self-assemble macromolecules and how to recreate these in living cells must be achieved. Bacillus thuringiensis subsp. israelenesis’ (Bti) crystalline inclusions are of exceptional interest, since they naturally package a single protein into a crystalline inclusion through a life cycle process called sporulation. Compared to classical macromolecular crystallography that takes a plethora of variables to exhaustion can still yield no crystals. The laborious process could be prevented; however, by better understanding Bt and these crystalline inclusions’ cellular self-assembly process. Cry11Ba is a protein packed into these crystalline (Cry) inclusions and found to be one of the most toxic pesticidal proteins. These crystals are then ingested by their host and switch from their packaged toxin crystal to their inactive protoxin at the high pH within their gut. My interests have been in elucidating the macromolecular structure from in vivo produced crystals, further understanding the ambiguous mode of action to gain better perspective for other δ-endotoxins, and probing the self-assembly of the crystalline inclusions in vivo throughout the sporulation process. I have studied Cry11Ba with structural analysis, solubility & toxicity assays, mutational studies, and imaging capabilities developed in cryo-EM to successfully in solve a de novo structure via in vivo crystalline inclusions, charting the pH sensitivity of the crystals, identifying key residues for stability and toxicity, analyzed the monomeric and multimeric particles in alkaline environments to understand Cry11Ba’s mode of action, and visualized previously unobserved sporulation stages for Bti.

  • 3 supplemental videos
Cover page: Structural and Biochemical Studies of Cell-Grown Cry11Ba Insecticidal Protein Crystals
  • Article
  • Peer Reviewed
UCLA Previously Published Works (2023)
ALECT2 systemic amyloidosis is associated with deposition of the leukocyte cell-derived chemotaxin-2 (LECT2) protein in the form of fibrils. In ALECT2 amyloidosis, ALECT2 fibrils deposit in the glomerulus, resulting in renal failure. Patients lack effective treatment options outside of renal transplant or dialysis. The structure of globular LECT2 has been determined but structures of ALECT2 amyloid fibrils remain unknown. Using single-particle cryo-EM, we find that recombinant human LECT2 forms robust twisting fibrils with canonical amyloid features. ALECT2 fibrils contain two mating protofilaments spanning residues 55-75 of the LECT2 sequence. The geometry of the ALECT2 fibril displays features in line with other pathogenic amyloids. Its core is tightly packed and stabilized by both hydrophobic contacts and hydrogen-bonded uncharged polar residues. The robustness of ALECT2 fibril cores is illustrated by their resistance to denaturants and proteases. This ALECT2 fibril structure presents a potential new target for treatments against ALECT2 systemic amyloidosis.
    Cover page: Cryo-EM structure of a human LECT2 amyloid fibril reveals a network of polar ladders at its core.
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)
    Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
      Cover page: De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals
      • Article
      UCLA Previously Published Works (2021)
      Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan , respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo -grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
        Cover page: De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals
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