Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

Healthy humans can be a source of antibodies countering COVID-19.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Engineering an efficient and bright split Corynactis californica green fluorescent protein.

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein-protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

Rational design of antimicrobial peptides targeting Gram-negative bacteria.

Membrane-targeting host antimicrobial peptides (AMPs) can kill or inhibit the growth of Gram-negative bacteria. However, the evolution of resistance among microbes poses a substantial barrier to the long-term utility of the host AMPs. Combining experiment and molecular dynamics simulations, we show that terminal carboxyl capping enhances both membrane insertion and antibacterial activity of an AMP called P1. Furthermore, we show that a bacterial strain with evolved resistance to this peptide becomes susceptible to P1 variants with either backbone capping or lysine-to-arginine substitutions. Our results suggest that cocktails of closely related AMPs may be useful in overcoming evolved resistance.

Split green fluorescent protein as a modular binding partner for protein crystallization.

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP ß-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Experimental mapping of soluble protein domains using a hierarchical approach.

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

1H, 15N and 13C backbone resonance assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis.

We report here the backbone assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis. The backbone resonance assignments were determined based on triple-resonance experiments with uniformly [13C,15N]-labeled protein in LMPG detergent micelles.

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.