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1. Controlling and quantifying protein concentration in Escherichia coli

   https://doi.org/10.1002/pro.3637  

   Speer, Shannon L.; Guseman, Alex J.; Patteson, Jon B.; Ehrmann, Brandie M.; Pielak, Gary J. (May 2019, Protein Science)

   Abstract The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)™Escherichia colicells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mMupon induction with 0.4 mMisopropyl β‐d‐thiogalactoside. We discuss the results in terms of what can and cannot be learned from in‐cell protein NMR studies inE. coli. 

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2. Intra-FCY1: a novel system to identify mutations that cause protein misfolding

   https://doi.org/10.3389/fgene.2023.1198203  

   Quan, N; Eguchi, Y; Geiler-Samerotte, K (September 2023, Frontiers in Genetics)

   Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteinsin vivooften rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novelin vivosystem named Intra-FCY1that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] inSaccharomyces cerevisiae. The Intra-FCY1system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1to illuminate the relationship between protein sequence and stability. 

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3. CRISPR /Cas9‐based editing of NF‐YC4 promoters yields high‐protein rice and soybean

   https://doi.org/10.1111/nph.20141  

   Wang, Lei; O'Conner, Seth; Tanvir, Rezwan; Zheng, Wenguang; Cothron, Samuel; Towery, Katherine; Bi, Honghao; Ellison, Evan_E; Yang, Bing; Voytas, Daniel_F; et al (September 2024, New Phytologist)

   Summary Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts.Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs.Building upon our prior research demonstrating the protein‐boosting effects of the transcription factor NF‐YC4, we identified conserved motifs targeted by RAV and WRKY repressors in theNF‐YC4promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increasedNF‐YC4expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein.Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality. 

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4. Measuring how two proteins affect each other's net charge in a crowded environment

   https://doi.org/10.1002/pro.4092  

   Dashnaw, Chad M.; Koone, Jordan C.; Abdolvahabi, Alireza; Shaw, Bryan F. (May 2021, Protein Science)

   Abstract Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester‐Staudinger pairs) can be used to mimic crowding by linking two non‐interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine‐acyl “protein charge ladders” and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z_monomer = −0.43 ± 0.01) and α‐lactalbumin (Z_monomer = −4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = −0.04 ± 0.09 upon crowding by this pair (Z_dimer = −5.10 ± 0.07). These small values of ΔZare not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions. 

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5. The intracellular environment affects protein–protein interactions

   https://doi.org/10.1073/pnas.2019918118  

   Speer, Shannon L.; Zheng, Wenwen; Jiang, Xin; Chu, I-Te; Guseman, Alex J.; Liu, Maili; Pielak, Gary J.; Li, Conggang (March 2021, Proceedings of the National Academy of Sciences)

   Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer,Escherichia colicells andXenopus laevisoocytes. The complex is more stable in cells than in buffer and more stable in oocytes thanE. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded thanE. colicells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment. 

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