Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17–22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron co-precipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe2+ ions and which residues are important for the MAPs functions.

The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications.

A 10-residue, glycosaminoglycan-binding peptide, GBP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBP has been proven to enter cells through macropinocytosis. The GBP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBP internalization. The intracellular penetration of GBP leading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.

The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation.

Ethylene plays versatile functions in regulating plant physiology. Although the high affinity ethylene receptor and its downstream regulators have been identified, the molecular recognition of the receptor interacting domains remains to be established. It has been speculated that the cytoplasmic signaling of the ethylene receptor is a two-component regulatory system involving the conserved receiver domain (RD). Here, we report the NMR chemical shift assignments for RD from Arabidopsis thaliana ethylene receptor ETR1. Nearly complete backbone and side-chain assignments were achieved at pH 6.0 and 25 °C. The assignments and backbone dynamics revealed the secondary structure and showed that ETR1-RD is a monomer in solution. These results will make it possible to monitor downstream binding partners and elucidates our understanding of phosphotransfer in the plant two-component regulatory system in the ethylene signaling pathway.