Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging.

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.

Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition.

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.

A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements.

Hydrogen exchange-mass spectrometry (HX-MS) is widely promoted for its ability to detect subtle perturbations in protein structure, but such perturbations will result in small differences in HX. However, the detection limit of HX-MS has not been widely investigated, nor is there a useful approach for defining the detection limit of HX-MS measurements. In this work, we designed a well-characterized structural variant spiking model to investigate the detection limit of conventional peptide-based HX-MS. The detection limit was challenged by spiking small fractions of a structural variant (modeled using maltose binding protein W169G mutant) into a reference protein (wild-type maltose binding protein). As little as 5% of the structural variant could be detected. The small structural perturbation was not resolvable by far UV circular dichroism, differential scanning calorimetry, or size exclusion chromatography. Furthermore, we validated the ability of the hybrid statistical analysis approach, presented in a companion paper (10.1021/acs.analchem.9b01325), to reliably identify small, significant differences in HX-MS measurements. With our structural variant spiking model, we demonstrate a benchmarking approach for determining a detection limit of HX-MS for detection of changes in higher-order structure that might be encountered in protein structural comparability and similarity assessment applications.

Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach.

Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures of proteins. Typically, the data sets are large with many differential HX values corresponding to many peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant differences in HX-MS measurements, a statistical analysis approach is needed. In this work, we performed null HX-MS measurements (i.e., no meaningful differences) on maltose binding protein and infliximab, a monoclonal antibody, to evaluate the reliability of different statistical analysis approaches. Null measurements are useful for directly evaluating the risk (i.e., falsely classifying a difference as significant) and power (i.e., failing to classify a true difference as significant) associated with different statistical analysis approaches. With null measurements, we identified weaknesses in the approaches commonly used. Individual tests of significance were prone to false positives due to the problem of multiple comparisons. Incorporation of Bonferroni correction led to unacceptably large limits of detection, severely decreasing the power. Analysis methods using a globally estimated significance limit also led to an overestimation of the limit of detection, leading to a loss of power. Here, we demonstrate a hybrid statistical analysis, based on volcano plots, that combines individual significance testing with an estimated global significance limit, that simultaneously decreased the risk of false positives and retained superior power. Furthermore, we highlight the utility of null HX-MS measurements to explicitly evaluate the criteria used to classify a difference in HX as significant.

Enzyme-linked immunosorbent assay and agar gel immunodiffusion assay for diagnosis of equine infectious anemia employing p26 protein fused to the maltose-binding protein.

A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.

Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment.

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαß) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.

Discovery of highly reactive peptide tag by ELISA-type screening for specific cysteine conjugation.

We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.

Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor.

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer(2) (BRET(2)) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET(2) provides a better matched Förster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET(2) tagged MBP protein. The half maximal effective concentration (EC(50)) was 0.37 µM for maltose and the response was linear over almost three log units ranging from 10nM to 3.16 µM maltose for the BRET(2) system compared to an EC(50) of 2.3 µM and a linear response ranging from 0.3 µM to 21.1 µM for the equivalent FRET-based biosensor. The biosensor's estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET(2)-based transduction scheme approach would likely be applicable to other binding proteins that have a "venus-fly-trap" mechanism.

The importance of adding EDTA for the nanopore analysis of proteins.

Nanopore analysis is a promising technique for studying the conformation of proteins and protein/protein interactions. Two proteins (bacterial thioredoxin and maltose binding protein) were subjected to nanopore analysis with α-hemolysin. Two types of events were observed; bumping events with a blockade current less than -40 pA and intercalation events with blockade currents between -40 pA and -100 pA. In potassium phosphate buffer, pH 7.8, both proteins gave intercalation events but the frequency of these events was significantly reduced in TRIS or HEPES buffers especially in the presence of 0.01 mM divalent metal ions. The frequency of events was restored by the addition of EDTA. For maltose binding protein, the frequency of intercalation events was also decreased in the presence of maltose but not lactose to which it does not bind. It is proposed that the events with large blockade currents represent transient intercalation of a loop or end of the protein into the pore and that divalent metal ions inhibit this process. The results demonstrate that the choice of buffer and the effects of metal ion contamination are important considerations in nanopore analysis.