Effect of the Competing Ligand on Ni-NTA/Histag Strength Revealed by Click Chemistry-Based Force Spectroscopy.

The specific interaction between Ni-nitrilotriacetic acid and the six-histidine tag may be one of the most important coordination bonds utilized in biological research because of its wide application for recombinant protein purification. The complex stability is critical for target protein binding. Thus, measurement of the mechanical stability of the system was attempted soon after the invention of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protons are the two critical factors for target protein elution. However, the mechanochemistry between the system and the imidazole/proton has not been determined. Here, an AFM-SMFS system using strain-promoted alkyne-azide cycloaddition and Cu-free click chemistry was used to characterize the system. Consequently, the destabilizing effect of the imidazole and proton on the interaction was revealed quantitatively, leading to a 3-fold increase in the bond dissociation rate.

Single-Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions.

The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron-sulfur protein containing a unique Fe2 S2 (His)1 (Cys)3 metal cluster with a single Fe-N(His87) coordinating bond. This labile Fe-N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe-N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM-SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single-molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe-N bond.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy.

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene. It plays an essential role in many biological systems as a vital driving force for protein folding, stability, and receptor-ligand interaction/recognition. To date, the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional (3D) protein structures and corresponding computational calculations. However, their experimental verification and quantification remain sparse at the molecular level, mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins. Here, we use atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of protein neutrophil gelatinase-associated lipocalin (also known as NGAL, siderocalin, lipocalin 2) that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols. Based on a site-specific cysteine engineering and anchoring method, we first characterized the stability and unfolding pathways of apo-NGAL. Then, the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized. AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites, F122 and F133. Here, NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect. Thus, our work demonstrates high-precision detection of the weak cation-π interaction in NGAL. Electronic Supplementary Material: Supplementary material (additional SDS-PAGE, UV-vis, protein sequences, and more experimental methods) is available in the online version of this article at 10.1007/s12274-021-4065-9.

Transforming de novo protein α3D into a mechanically stable protein by zinc binding.

α3D is a de novo designed three-helix bundle protein. Like most naturally occurring helical proteins, it is mechanically labile with an unfolding force of <15 pN, revealed by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). This protein has been further designed with a tri-cysteine metal-binding site, named α3DIV, which can bind heavy transition metals. Here, we demonstrate that incorporating such a metal-binding site can transform this mechanically labile protein into a stable one. We show that zinc binds to the tri-cysteine site and increases the unfolding force to ∼160 pN. This force is one order of magnitude higher than that of the apo-protein (<15 pN). Moreover, the unfolding mechanism of Zn-α3DIV indicates the correct zinc binding with the tri-cysteine site, forming three mechanostable Zn-thiolate bonds. Thus, α3DIV could be a potential α-helical structure-based building block for synthesizing biomaterials with tunable mechanical properties.

Expression profile analysis of a new testis-specifically expressed gene C17ORF64 and its association with cell apoptosis in MCF-7 cells.

With the increasing incidence of male infertility, identification and investigation the functions of new genes related to spermatogenesis are effective avenues to elucidate the decline of testicular function. In this study, a new gene, C17ORF64 (chromosome 17 open reading frame 64), was identified from mouse testes and its potential function was studied.RT-PCR and qRT-PCR assay showed that C17ORF64 mRNA was expressed exclusively in mouse testes and up-regulated from the 3-week old to 6-month old testes during postpartum development, which is consistent with C17ORF64 protein expression profile by western blotting analysis. Immunohistochemical analysis revealed that C17ORF64 protein was mainly localized in the cytoplasm of spermatogonia and spermatocytes, which is verified by GFP- labeled C17ORF64 gene expressed in GC-1 cells. C17ORF64 overexpression not only promoted cell apoptosis in MCF-7 cells, but also significantly decreased cell viability via MTT assay. Flow cytometric assay showed that C17ORF64 overexpression could inhibit cell cycle progression by arresting G1/S transition. Western blot and qRT-PCR analysis revealed that C17ORF64 overexpression inhibited the expression of anti-apoptotic protein bcl-2 and increased the expressions of pro-apoptotic protein caspase-3, caspase-8, caspase-9, Bax, P21 and P53. Taken together, our results confirmed C17ORF64 testis-specific expression pattern and, for the first time, demonstrated that C17ORF64 could inhibit cell viability and accelerate apoptosis in MCF-7 cells through caspase-3 regulatory pathways.

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

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.

Single-Molecule Force Spectroscopy Reveals that Iron-Ligand Bonds Modulate Proteins in Different Modes.

The iron-amino acid interactions Fe-O(Glu/Asp), Fe-N(His), and Fe-S(Cys) are the three major iron-ligand bonds in proteins. To compare their properties in proteins, we used atomic force microscopy (AFM)-based single-molecule force spectroscopy to investigate a superoxide reductase (Fe(III)-SOR) with all three types of bonds forming an Fe(His)4CysGlu center. We first found that Apo-SOR without bound iron showed multiple unfolding pathways only from the ß-barrel core. Then, using Holo-SOR with a ferric ion, we found that a single Fe-O(Glu) bond can tightly connect the flexible N-terminal fragment to the ß-barrel and stabilize the whole protein, showing a complete protein unfolding scenario, while the single Fe-N(His) bond was weak and unable to provide such a stabilization. Moreover, when multiple Fe-N bonds are present, a similar stabilization effect can be achieved. Our results showed that the iron-ligand bond modulates protein structure and stability in different modes at the single-bond level.