Education & Certifications


  • Phd, Washington State University (2020)

All Publications


  • Three-dimensional structure-guided evolution of a ribosome with tethered subunits. Nature chemical biology Kim, D. S., Watkins, A., Bidstrup, E., Lee, J., Topkar, V., Kofman, C., Schwarz, K. J., Liu, Y., Pintilie, G., Roney, E., Das, R., Jewett, M. C. 2022

    Abstract

    RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

    View details for DOI 10.1038/s41589-022-01064-w

    View details for PubMedID 35836020

  • Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element-specific proteins reveal differentiation of the endomembrane system. Proceedings of the National Academy of Sciences of the United States of America Liu, Y., Vasina, V. V., Kraner, M. E., Peters, W. S., Sonnewald, U., Knoblauch, M. 2022; 119 (1)

    Abstract

    Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

    View details for DOI 10.1073/pnas.2112755119

    View details for PubMedID 34983847

  • Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness PLANT AND CELL PHYSIOLOGY Liu, Y., Peters, W. S., Froelich, D. R., Howell, A. H., Mooney, S., Evans, J. E., Hellmann, H. A., Knoblauch, M. 2020; 61 (10): 1699-1710

    Abstract

    Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

    View details for DOI 10.1093/pcp/pcaa093

    View details for Web of Science ID 000607173600003

    View details for PubMedID 33035344

  • Protein structural biology using cell-free platform from wheat germ ADVANCED STRUCTURAL AND CHEMICAL IMAGING Novikova, I. V., Sharma, N., Moser, T., Sontag, R., Liu, Y., Collazo, M. J., Cascio, D., Shokuhfar, T., Hellmann, H., Knoblauch, M., Evans, J. E. 2018; 4: 13

    Abstract

    One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (>‚ÄČ98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.

    View details for DOI 10.1186/s40679-018-0062-9

    View details for Web of Science ID 000449823300001

    View details for PubMedID 30524935

    View details for PubMedCentralID PMC6244559