The molecular sieving properties of protein surface-attached polymers are the central features in how polymers extend therapeutic protein lifetimes in vivo. Yet, even after 30 years of research, permeation rates of molecules through polymersurrounded protein surfaces are largely unknown. As a result, the generation of protein−polymer conjugates remains a stochastic process, unfacilitated by knowledge of structure-function-polymer architecture relationships. In this work, polymers are grown from the surface of avidin using atom transfer radical polymerization (ATRP) and used to determine how polymer length and density influence the binding kinetics of ligands as a function of ligand size and shape. The rate of binding is strongly dependent on the grafting density of polymers and the size of the ligand but interestingly, far less dependent on the length of the polymer. This study unveils a deeper understanding of relationship between polymer characteristics and binding kinetics, discovering important steps in rational design of protein−polymer conjugates.

The spliceosome is a dynamic macromolecular machine that undergoes a series of conformational rearrangements as it transitions between the several states required for accurate splicing. The transition from the B to B is a key part of spliceosome assembly and is defined by the departure of several proteins, including essential U5 component Dib1. Recent structural studies suggest that Dib1 has a role in preventing premature spliceosome activation, as it is positioned adjacent to the U6 snRNA ACAGAGA and the U5 loop I, but its mechanism is unknown. Our data indicate that Dib1 is a robust protein that tolerates incorporation of many mutations, even at positions thought to be key for its folding stability. However, we have identified two temperature-sensitive mutants that stall in vitro splicing prior to the first catalytic step and block assembly at the B complex. In addition, Dib1 readily exchanges in splicing extracts despite being a central component of the U5 snRNP, suggesting that the binding site of Dib1 is flexible. Structural analyses show that the overall conformation of Dib1 and the mutants are not affected by temperature, so the temperature sensitive defects most likely result from altered interactions between Dib1 and other spliceosomal components. Together, these data lead to a new understanding of Dib1's role in the B to B transition and provide a model for how dynamic protein-RNA interactions contribute to the correct assembly of a complex molecular machine.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

After labeling of proteins with chemical tags, the unconjugated tag that remains in solution must be removed to prevent interference with downstream workflows. Currently, unconjugated tags are removed using dialysis or spin filtration, which suffers from significant sample loss due to protein precipitation, transfer steps, and lengthy protocols. In this study, polymer-based protein engineering was used to sequester the small molecule biotin. By enclosing avidin in a polymer cage, known as caged-avidin, free, unconjugated biotin was able to bind to avidin’s active site, while more than 90% of biotinylated proteins were excluded. In the presence of excess biotin, less than 10% of biotinylated protein bound to avidin-coated beads (known as NeutrAvidin) due to competition from the free biotin. However, sequestration of the excess free biotin by caged-avidin allowed recovery of up to 93% binding of biotinylated proteins to NeutrAvidin beads. Utilization of caged-avidin enabled the use of biotin-CDM, a reversible biotin tag, for tagging, capture, cleanup, and release of a single protein sample and a whole yeast proteome, improving the final protein yields to 97%. This study demonstrates an application for the sieving effect of polymers on the surface of proteins to sequester and remove small molecules from solution without dialysis.

The spliceosome is a highly dynamic molecular machine composed of small nuclear ribonucleoprotein particles (snRNP). The snRNP facilitates the joining of exons via two transesterification reactions that remove the non‐coding intronic regions. Once the introns are removed, the resulting mature RNA (mRNA) transcript can then be translated into functional protein. The fidelity of this process is crucial, and mistakes in splicing can lead to diseases such as Retinitis pigmentosa. One of the critical steps in splicing is the activation of the U4/U6‐U5 triple snRNP upon joining the spliceosomal complex.­­ In this study, we focus on the small, essential splicing protein Dib1, whose function in splicing remains unknown. Dib1 is 15kDa protein with a thioredoxin‐like fold. Previous data suggests that Dib1 interacts with splicing proteins that bridge the U4/U6 and U5 snRNPs. We characterized the role of Dib1 by rationally designing dib1 mutants and assaying for effects on Saccharomyces cerevisiae growth. We purified these Dib1 mutant proteins and examined their structures by circular dichroism in order to determine the effects these mutants have on the structure of Dib1 protein. In addition, splicing assays were conducted in order to determine the effects the mutant may have on mediating splicing. These assays will help further characterize Dib1 and its role in splicing as well as lend insight into further understanding the splicing machinery.

Many bacterial hosts in recent years have been shown to have a profound effect on host behavior, and the endosymbiotic alphaproteobacteria Wolbachia is no exception. Present in at least 40% of the insect population, the maternally transmitted Wolbachia modulates behaviors such as male aggression, mate selection, and migration. Wolbachia have also been shown to change host response to olfactory cues, influence life span, and prevent infection by certain pathogens. This last feature of Wolbachia, specifically the prevention of infection by viral pathogens such as Dengue, Zika, and Chikungunya in mosquitos, has become of recent interest as a potential mechanism to suppress transmission of these disease‐causing viruses to humans.To tease apart the mechanisms by which Wolbachia influences host behavior and viral suppression, Drosophila, a genetically tractable model organism that is commonly used to delve into the molecular and cell biology of the symbiont behavioral, was used. A proteomic screen of changes in the Drosophila brain upon Wolbachia infection was performed using 2D Differential Gel Electrophoresis and LC‐MS. Protein abundance changes and post translational modification changes were observed and compared in both Drosophila simulans and Drosophila melanogaster as well as between male and female populations. Post translational modification changes were observed in multiple metabolic proteins and proteins responsible for neurotransmitter synthesis. This study describes the protein and post translational changes identified after Wolbachia infection and how they might contribute to behavioral changes in the host.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.Support or Funding InformationThis work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autoimmune diseases affect >20 million people in the US today. Currently, disease‐specific autoantibodies are thought to be the best biomarkers for diagnosis. Conventional immunoprecipitation methods have been used to identify autoantigens from the most common autoimmune diseases. However, these diseases account for only 6.5 million of the 20 million patients suffering from autoimmune diseases, leaving many without diagnoses until irreversible damage occurs. The remaining 13.5 million patients have >70 autoimmune disorders without well characterized autoantibodies. The state‐of‐the‐art diagnostic test of these remaining diseases relies on gel electrophoresis of immunoprecipitated radiolabeled proteins, which cannot be identified by MS due to safety issues and the overwhelming presence of immunoglobulins. We have created an immunoprecipitation method that uses serum from patients with any autoimmune disorder to identify patient‐specific autoantigen proteins. This method uses a reversible click chemistry tag, called ProMTag. One end of the ProMTag forms a reversible, covalent bond with protein by coupling to lysines and amino termini. The other end of the ProMTag can form an irreversible, covalent bond with a solid bead support via a click chemistry, methyltetrazine‐TCO, pairing. In this study, the proteins of cell lysates that contain potential autoantigens were labeled with ProMTag. The ProMTagged‐proteins were exposed to patient antibodies bound to Protein A beads, thus capturing the ProMTagged autoantigens. All proteins were released from the Protein A beads, including ProMTagged‐autoantigens and untagged‐antibodies. The ProMTagged‐autoantigens were subsequently coupled to TCO beads, and the untagged‐antibodies were washed away. The linkage between the ProMTag and autoantigens was then reversed, yielding autoantigen proteins with greatly reduced antibody contamination ready for MS analysis. MS analysis successfully identified autoantigens from patient serums with rheumatoid arthri. This autoimmune biomarker discovery method can accelerate sample testing for known autoantigens and facilitating rapid discovery of novel autoantigens for both diagnostic and predictive biomarkers.