Improved structure and enhanced the insecticidal activity of Sip1Aa protein by adding disulfide bonds

Sip1Aa is an insecticidal protein of Bacillus thuringiensis at the secretory stage. It has a strong toxic effect on the members of order Coleoptera. To date, there are few available studies on Sip1Aa protein and the inclusion body problem is serious, and this raises the importance to conduct further studies on Sip1Aa protein. Disulfide bonds, as the only covalent bond on protein side chains, play an important role in the stability and function of the proteins. The tertiary structure of Sip1Aa protein was analyzed by homologous modeling and other bioinformatics methods to predict the conserved domain of Sip1Aa protein. Cysteine used to replace these amino acids by site-directed mutation. Consequently, we were able to successfully construct Sip149-251, Sip153-248, Sip158-243, and Sip178-314. These were exposed to ultraviolet radiation, and we found that Sip153-248 and Sip158-243 were the most stable, followed by Sip149-251 and Sip178-314 when compared with Sip1Aa. After the mutant strain was transferred into Escherichia coli BL21, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect the inducible expression products. Approximately 37.6 kDa of proteins that were highly expressed in E. coli. We found no significant change in the insecticidal activity.

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

Identification of allosteric disulfides from labile bonds in X-ray structures

Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecules of a protein crystal but absent in others, or present in some structures of a protein but absent in others. We reasoned that the labile nature of these disulfides signifies a propensity for cleavage and so possible allosteric regulation of the protein in which the bond resides. A total of 511 labile disulfide bonds were identified. The labile disulfides are more stressed than the average bond, being characterized by high average torsional strain and stretching of the sulfur–sulfur bond and neighbouring bond angles. This pre-stress likely underpins their susceptibility to cleavage. The coagulation, complement and oxygen-sensing hypoxia inducible factor-1 pathways, which are known or have been suggested to be regulated by allosteric disulfides, are enriched in proteins containing labile disulfides. The identification of labile disulfide bonds will facilitate the study of this post-translational modification.

A Knot Polynomial Invariant for Analysis of Topology of RNA Stems and Protein Disulfide Bonds

Knot polynomials have been used to detect and classify knots in biomolecules. Computation of knot polynomials in DNA and protein molecules have revealed the existence of knotted structures, and provided important insight into their topological structures. However, conventional knot polynomials are not well suited to study RNA molecules, as RNA structures are determined by stem regions which are not taken into account in conventional knot polynomials. In this study, we develop a new class of knot polynomials specifically designed to study RNA molecules, which considers stem regions. We demonstrate that our knot polynomials have direct structural relation with RNA molecules, and can be used to classify the topology of RNA secondary structures. Furthermore, we point out that these knot polynomials can be used to model the topological effects of disulfide bonds in protein molecules.