We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr), at the N or C-terminus of a peptide or protein, and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction by 220-fold, enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and trastuzumab antibody. This study demonstrates short peptide tags could aid in accelerating bond forming reactions that are often slow to non-existent in water.
A "D-scan" of two small proteins, the disulfide-rich Ecballium elaterium trypsin inhibitor II (EETI-II) and a minimized Z domain of protein A (Z33), is reported. For each protein, the stereochemistry of one amino acid at a time was inverted to generate a series of diastereomers. In much the same way an alanine scan determines necessary residues for protein function, the D-scan elucidated the critical stereocenters of the 30-residue EETI-II and the 33-residue Z33. The folding properties and activity of each variant were investigated. A total of 24 out of 30 EETI-II D-scan analogues folded to give a three-disulfide product. Of the 24 variants that folded, half were high-affinity trypsin inhibitors, and three were as active as the wild type (WT). Of these 12 active variants, most were substantially less stable to reduction than WT EETI-II (WT first reduction potential -270.0 ± 1.5 mV, WT second reduction potential -307.2 ± 1.1 mV). Similarly, ten Z33 analogues retained high binding affinity to IgG (KD < 250 nM, WT: 24 ± 1 nM) and 12 additional analogues had reduced but appreciable IgG binding affinity (KD between 250 nM and 2.5 μM). As with EETI-II, most Z33 analogues were substantially less stable than the WT (ΔG(H2O, 263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show that the D-scan is powerful new strategy for studying how the stereochemistry of amino acids affects the structure and function of proteins.
Transient currents in the solar wind are carried by various magnetic field discontinuities that contribute significantly to the magnetic field fluctuation spectrum. Internal instabilities and dynamics of these discontinuities are believed to be responsible for magnetic field energy dissipation and corresponding charged particle acceleration and heating. Accurate modeling of these phenomena requires detailed investigation of transient current formation and evolution. By examining such evolution using a unique data set compiled from observations of the same solar wind flow by two spacecraft at Earth’s and Mars’s orbits, we show that it consists of several processes: discontinuity thinning (decrease in thickness normalized by the ion inertial length), intensification of currents normalized to the proton thermal current (i.e., the product of proton charge, density, and thermal velocity), and increase in the compressional component of magnetic field variations across discontinuities. The significant proton temperature variation around most observed discontinuities indicates possible proton heating. Plasma velocity jumps across the discontinuities are well correlated with Alfvén velocity changes. We discuss possible explanations of the observed discontinuity evolution. We also compare the observed evolution with predictions of models describing discontinuity formation due to Alfvén wave steepening. Our results show that discontinuity modeling likely requires taking into account both the effects of nonlinear Alfvén wave dynamics and solar wind expansion.
Magnetic flux ropes (MFR) are universal magnetoplasma structures (similar to cylindrical screw pinches) formed in reconnecting current sheets. In particular, MFR with scales from about the ion inertial length to MHD range are widely observed in the Earth magnetosphere. Typical MFR have force-free configuration with the axial magnetic field peaking on the MFR axis, whereas bifurcated MFR with an off-axis peak of the axial magnetic field are observed as well. In the present paper, we develop kinetic models of force-free and bifurcated MFR and determine consistent ion and electron distribution functions. The magnetic field configuration of the force-free MFR represents well-known Gold-Hoyle MFR (uniformly twisted MFR). We show that bifurcated MFR are characterized by the presence of cold and hot current-carrying electrons. The developed models are capable to describe MFR observed in the Earth magnetotail as well as MFR recently observed by Magnetospheric Multiscale Mission at the Earth magnetopause.
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