Biology subjects

Kuru, E.

Publications and source records attributed to Kuru, E..

10 recordsLinked to original sources

bioRxiv2026

Genetic code expansion enables programmable covalent protein design

Covalent chemistry has transformed small-molecule drug discovery, yet analogous strategies for proteins remain largely inaccessible because covalent warheads cannot be readily integrated into biologics. Conventional genetic code expansion requires engineering a dedicated aminoacyl-tRNA synthetase for each new amino acid, rendering broad warhead screening impractical. Here we introduce AminoX, a platform that bypasses this limitation through direct tRNA acylation, enabling site-specific incorporation of chemically diverse non-standard amino acids (nsAAs), including covalent warhead nsAAs compatible with scalable biologic manufacturing and multifunctional nsAAs. Using a pooled mRNA display workflow, we screened more than 2,000 warhead-position combinations in machine learning-designed de novo miniproteins targeting CTLA-4, enabling parallel interrogation of covalent chemistry, linker geometry, and incorporation site. We confirmed covalent engagement on cells together with enhanced functional blockade. Finally, we demonstrate multifunctional nsAAs that combine covalent warheads with fluorogenic reporters for real-time detection of target engagement, as well as dual nsAA incorporation for macrocyclization and fluorescent imaging of covalent binding on cell surfaces. By uniting synthetic biology, chemical biology, generative protein design, and high-throughput functional selection, AminoX compresses covalent protein engineering timelines by orders of magnitude, accelerating the development of next-generation therapeutics, biosensors, and chemical probes.

synthetic biology↗

bioRxiv2025

The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis. These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan (AG). They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in Corynebacterium glutamicum (Cglu) using PorH as a model corynebacterial MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu coordinates outer membrane protein assembly with PG biogenesis and uses proteins to connect the mycomembrane and the cell wall.

microbiology↗

bioRxiv2024

Atomistic Mechanisms of Calcium Permeation Modulated by Q/R Editing and Selectivity Filter Mutations in GluA2 AMPA Receptors

GluA2 is a key subunit of AMPA receptor ion channels that is abundantly expressed in the vertebrate brain. Post-transcriptional Q/R editing of GluA2 renders AMPARs nearly impermeable to calcium ions, which is crucial for their normal function. Although previous studies have characterized conductivity and selectivity differences between edited and unedited GluA2 variants and heteromeric receptors incorporating GluA2, the atomistic mechanism remains largely unknown. In this study, we investigate ion permeation in the context of multiple Ca2+ binding sites along the pore predicted from MD simulations, considering both mutations and co-permeating monovalent ions. Patch clamp electrophysiology recordings confirmed a binding site at the intracellular mouth of the selectivity filter that confers selectivity for calcium over monovalent ions. A patient mutation at the same site has been previously shown to cause neurodevelopmental abnormalities. Furthermore, MD simulations of GluA2 with different arginine copy number at the Q/R site show that Ca2+ conduction is blocked in the presence of two arginines, whereas K+ is only blocked by four arginines, in explaining the results from decades of electrophysiological work. Finally, MD simulations revealed that Ca2+ reduces K+ conduction by preferentially occupying the intracellular SF binding site, whereas Na+ does not. This result is consistent with electrophysiological results from the D590 mutants, and suggests that divalent binding in the selectivity filter is a major determinant of AMPAR conductance.

biophysics↗

bioRxiv2024

Preventing escape and malfunction of recoded cells due to tRNA base changes

Engineering the genetic code restricts DNA transfer (cellular bioisolation) and enables new chemistries via non-standard amino acid incorporation. These distinct properties make recoded cells state-of-the-art safe technologies. However, evolutionary pressures may endanger the longevity of the recoding. Here, we reveal that recoded Escherichia coli lacking 18,214 serine codons and two tRNASer can express wild-type antibiotic resistance genes and escape up to seven orders of magnitude faster than expected. We show a two-step escape process whereby recoded cells mistranslate antibiotic resistance genes to survive until modified or mutated tRNAs reintroduce serine into unassigned codons. We developed genetic-code-sensitive kill switches that sense serine incorporation and prevent cellular escape while preserving encoding of three distinct non-standard amino acids. This work lays the foundation for the long-term controlled function of cells that incorporate new chemistries, with implications for the design, use, and biosafety of synthetic genomes in clinical and environmental applications where physical containment is insufficient.

synthetic biology↗

bioRxiv2023

A Platform for Controlled Template-Independent Enzymatic Synthesis of RNA Oligonucleotides and Therapeutics

Therapeutic RNA oligonucleotides have shown tremendous potential to manage and treat disease, yet current manufacturing methods cannot deliver on this promise. Here, we report the development and optimization of a novel, aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methodologies. Our platform is made possible by reversible terminator nucleoside triphosphates and an enzyme capable of their incorporation. We show that many common therapeutic RNA modifications are compatible with our process and demonstrate the enzymatic synthesis of natural and modified oligonucleotides in both liquid and solid phases. Our platform offers many unique advantages over chemical synthesis, including the realization of a more sustainable process to produce therapeutic RNA oligonucleotides. One-Sentence SummaryAn enzyme and novel nucleotide building blocks are used to synthesize RNA oligonucleotides template independently under aqueous conditions.

synthetic biology↗

bioRxiv2021

Unipolar peptidoglycan synthesis in the Rhizobiales requires an essential class A penicillin-binding protein

Members of the Rhizobiales are polarly-growing bacteria that lack homologs of the canonical rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant-pathogen Agrobacterium tumefaciens. The development of fluorescent O_SCPLOWDC_SCPLOW-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis convincingly demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an alternative mode of cell wall synthesis that likely requires O_SCPLOWLDC_SCPLOW-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.

microbiology↗

bioRxiv2021

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Bacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

synthetic biology↗

bioRxiv2019

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. We show that the helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod bacteria. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

microbiology↗

bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed \"Post-Translational Proofreading\" (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

synthetic biology↗

bioRxiv2017

Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared to the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces ~4-fold more total protein. However the majority of these polypeptides are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the S30 crude extract system. Both of the two systems suffer from low ribosome recycling efficiency when translating proteins from 90 kD to 220 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination prior to energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while accumulation of inorganic phosphate contributes to reaction termination. Additionally, while adding EF-P to PURE reduced total protein translated, it also increased the fraction of active product and reduced partial translated product probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.

bioengineering↗