Native Structure
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2022 ◽  
Junjie Xiong ◽  
Han Wang ◽  
Xingzi Lan ◽  
Yaqi Wang ◽  
Zixu Wang ◽  

Abstract Many strategies have been adopted to engineer bone-ligament interface, which is of great value to both the tissue regeneration and the mechanism understanding underlying interface regeneration. However, how to recapitulate the complexity and heterogeneity of the native bone-ligament interface including the structural, cellular and mechanical gradients is still challenging. In this work, a bioinspired grid-crimp micropattern fabricated by melt electrospinning writing (MEW) was proposed to mimic the native structure of bone-ligament interface. The printing strategy of crimped fiber micropattern was developed and the processing parameters were optimized, which were used to mimic the crimp structure of the collagen fibrils in ligament. The guidance effect of the crimp angle and fiber spacing on the orientation of fibroblasts was studied, and both of them showed different levels of cell alignment effect.. MEW grid micropatterns with different fiber spacings were fabricated as bone region. Both the alkaling phosphatase activity and calcium mineralization results demonstrated the higher osteoinductive ability of the MEW grid structures, especially for that with smaller fiber spacing. The combined grid-crimp micropatterns were applied for the co-culture of fibroblasts and osteoblasts. The results showed that more cells were observed to migrate into the in-between interface region for the pattern with smaller fiber spacing, suggested the faster migration speed of cells. Finally, a cylindrical triphasic scaffold was successfully generated by rolling the grid-crimp micropatterns up, showing both structural and mechanical similarity to the native bone-ligament interface. In summary, the proposed strategy is reliable to fabricate grid-crimp triphasic micropatterns with controllable structural parameters to mimic the native bone-to-ligament structure, and the generated 3D scaffold shows great potential for the further bone-ligament interface tissue engineering.

2022 ◽  
Daisuke Fujinami ◽  
Seiichiro Hayashi ◽  
Daisuke Kohda

Multiprobe measurements, such as NMR and hydrogen exchange study, can provide the equilibrium constant K and kinetic rate constant k of the structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between residue-specific log K values and residue-specific log k values for the two-state topological isomerization of a 27-residue peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific equilibrium and kinetic constants in various exchange processes, including protein folding, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of log-log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Protein molecules quickly fold into their native structures and change their conformations smoothly. Theoretical studies and molecular simulations advocate that the physicochemical basis is the consistency principle and the minimal frustration principle: Non-native structures/interactions are absent or minimized along the folding pathway. The linearity of the residue-based free energy relationship demonstrates experimentally the absence of non-native structures in transition states. In this context, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the non-native structure, which had been identified by other experiments. The rbLFER provides a unique and practical method to probe the dynamic aspects of the transition states of protein molecules.

Pharmaceutics ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 71
Itzik Cooper ◽  
Michal Schnaider-Beeri ◽  
Mati Fridkin ◽  
Yoram Shechter

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

2021 ◽  
Anirban Das ◽  
Yogesh Gangarde ◽  
Ishu Saraogi

Insulin, a peptide hormone, is susceptible to amyloid formation upon exposure to aberrant physiological conditions, result-ing in a loss of its bioactivity. For mitigating insulin aggregation, we report a molecule called PAD-S, which completely inhibit-ed insulin fibril formation, and preserved insulin in its soluble form. Circular Dichroism spectroscopy showed that PAD-S was able to maintain the native structure of insulin, thus acting as a chemical chaperone. Seeded aggregation kinetics suggest that PAD-S inhibited primary nucleation events during aggregation. This is consistent with molecular docking results which suggest that PAD-S binds strongly to native insulin monomers/dimers. Through a competitive binding experiment with ‘LVEALYL’ peptide, we conclude that PAD-S likely binds to the amyloid prone B11-B17 residues of insulin thereby prevent-ing its aggregation. PAD-S was also effective in disaggregating preformed insulin fibrils to non-toxic species. PAD-S treated insulin was functional as indicated by its ability to phosphorylate Akt. PAD-S was also highly effective in preventing the ag-gregation of insulin biosimilars. The low cellular cytotoxicity of PAD-S, and amelioration of aggregation-induced toxicity by PAD-S treated insulin further highlights its potential as an effective chemical chaperone.

Polymers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 4134
Yuliya Nashchekina ◽  
Pavel Nikonov ◽  
Nataliya Mikhailova ◽  
Alexey Nashchekin

Collagen in the body is exposed to a range of influences, including free radicals, which can lead to a significant change in its structure. Modeling such an effect on collagen fibrils will allow one to get a native structure in vitro, which is important for modern tissue engineering. The aim of this work is to study the effect of free radicals on a solution of hydrogen peroxide with a concentration of 0.006–0.15% on the structure of collagen fibrils in vitro, and the response of cells to such treatment. SEM measurements show a decrease in the diameter of the collagen fibrils with an increase in the concentration of hydrogen peroxide. Such treatment also leads to an increase in the wetting angle of the collagen surface. Fourier transform infrared spectroscopy demonstrates a decrease in the signal with wave number 1084 cm−1 due to the detachment of glucose and galactose linked to hydroxylysine, connected to the collagen molecule through the -C-O-C- group. During the first day of cultivating ASCs, MG-63, and A-431 cells, an increase in cell adhesion on collagen fibrils treated with H2O2 (0.015, 0.03%) was observed. Thus the effect of H2O2 on biologically relevant extracellular matrices for the formation of collagen scaffolds was shown.

2021 ◽  
Kayla Gentile ◽  
Ashlesha Bhide ◽  
Joshua Kauffman ◽  
Subhadip Ghosh ◽  
Subhabrata Maiti ◽  

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-Glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

2021 ◽  
John D. MacMicking ◽  
Shiwei Zhu ◽  
Clinton J Bradfield ◽  
Agnieszka Maminska ◽  
Euisoon Park ◽  

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, these activities generate large supramolecular complexes that recruit immune proteins for protection. Here, we solve the native structure of a massive antimicrobial complex generated by polymerization of 30,000 human guanylate-binding proteins (GBPs) over the entire surface of virulent bacteria. Construction of this giant nanomachine takes ~1-3 minutes, remains stable for hours, and acts as a cytokine and cell death signaling platform atop the coated bacterium. Cryo-ET of this coatomer revealed thousands of human GBP1 molecules undergo ~260 Angstrom insertion into the bacterial outer membrane, triggering lipopolysaccharide release that activates co-assembled caspase-4. Together, our results provide a quasi-atomic view of how the GBP coatomer mobilizes cytosolic immunity to combat infection in humans.

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256946
Yukiko Ohara ◽  
Yuriko Ozeki ◽  
Yoshitaka Tateishi ◽  
Tsukasa Mashima ◽  
Fumio Arisaka ◽  

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