Jingyuan Nie

Bio: Jingyuan Nie is an academic researcher from Nanjing University. The author has contributed to research in topics: Force spectroscopy & Protein domain. The author has an hindex of 1, co-authored 3 publications receiving 2 citations.

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy.

Abstract: Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Exploration of Metal-Ligand Coordination Bonds in Proteins by Single-molecule Force Spectroscopy

Abstract: Thanks to the binding of various metal ions, metalloprotein plays an essential role in many different biological processes and represents an indispensable protein subgroup. Thus, the knowledge of t...

Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells.

Abstract: Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for ∼120 days, are biocompatible, and are immunologically largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clinical trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiological conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.

The Local Vibrational Mode Theory and Its Place in the Vibrational Spectroscopy Arena.

Abstract: This Feature Article starts highlighting some recent experimental and theoretical advances in the field of IR and Raman spectroscopy, giving a taste of the breadth and dynamics of this striving field. The local mode theory is then reviewed, showing how local vibrational modes are derived from fundamental normal modes. New features are introduced that add to current theoretical efforts: (i) a unique measure of bond strength based on local mode force constants ranging from bonding in single molecules in different environments to bonding in periodic systems and crystals and (ii) a new way to interpret vibrational spectra by pinpointing and probing interactions between particular bond stretching contributions to the normal modes. All of this represents a means to work around the very nature of normal modes, namely that the vibrational motions in polyatomic molecules are delocalized. Three current focus points of the local mode analysis are reported, demonstrating how the local mode analysis extracts important information hidden in vibrational spectroscopy data supporting current experiments: (i) metal-ligand bonding in heme proteins, such as myoglobin and neuroglobin; (ii) disentanglement of DNA normal modes; and (iii) hydrogen bonding in water clusters and ice. Finally, the use of the local mode analysis by other research groups is summarized. Our vision is that in the future local mode analysis will be routinely applied by the community and that this Feature Article serves as an incubator for future collaborations between experiment and theory.

Direct Measurements of the Cobalt‐Thiolate Bonds Strength in Rubredoxin by Single‐Molecule Force Spectroscopy

Abstract: Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt‐containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal‐binding proteins through the formation of cobalt‐ligand bonds. Since most of these natural and artificial cobalt‐containing proteins are stable, we believe that these cobalt‐ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS) to directly measure the rupture force of Co(II)‐thiolate bond in Co‐substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling‐based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)n suitable for single‐molecule studies. Thus, we quantified the strength of Co(II)‐thiolate bonds in rubredoxin with a rupture force of ∼140 pN, revealing that it is a mechanostable chemical bond. In addition, the Co−S bond is more labile than the Zn−S bond in proteins, similar to the result from the metal‐competing titration experiment.

Single-Molecule Force Spectroscopy Reveals the Dynamic HgS Coordination Site in the De Novo-Designed Metalloprotein α3DIV.

Abstract: The de novo-designed metalloprotein α3DIV binds to mercury via three cysteine residues under dynamic conditions. An unusual trigonal three-coordinate HgS3 site is formed in the protein in basic solution, whereas a linear two-coordinate HgS2 site is formed in acidic solution. Furthermore, it is unknown whether the two coordinated cysteines in the HgS2 site are fixed or not, which may lead to more dynamics. However, the signal for HgS2 sites with different cysteines may be similar or may be averaged and indistinguishable. To circumvent this problem, we adopt a single-molecule approach to study one mercury site at a time. Using atomic force microscopy-based single-molecule force spectroscopy, the protein is unfolded, and the HgS site is ruptured. The results confirm the formation of HgS3 and HgS2 sites at different pH values. Moreover, it is found that any two of the three cysteines in the protein bind to mercury in the HgS2 site.

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths.

Abstract: The synthesis of ultra-small gold nanoclusters (Au NCs) with sizes down to 2 nm has received increasing interest due to their unique optical and electronic properties. Like many peptide-coated gold nanospheres synthesized before, modified gold nanoclusters with peptide conjugation are potentially significant in biomedical and catalytic fields. Here, we explore whether such small-sized gold nanoclusters can be conjugated with peptides also and characterize them using atomic force microscopy. Using a long and flexible elastin-like polypeptide (ELP)20 as the conjugated peptide, (ELP)20-Au NCs was successfully synthesized via a one-pot synthesis method. The unique optical and electronic properties of gold nanoclusters are still preserved, while a much larger size was obtained as expected due to the peptide conjugation. In addition, a short and rigid peptide (EAAAK)3 was conjugated to the gold nanoclusters. Their Yong's modulus was characterized using atomic force microscopy (AFM). Moreover, the coated peptide on the nanoclusters was pulled using AFM-based single molecule-force spectroscopy (SMFS), showing expected properties as one of the first force spectroscopy experiments on peptide-coated nanoclusters. Our results pave the way for further modification of nanoclusters based on the conjugated peptides and show a new method to characterize these materials using AFM-SMFS.