The Protein Journal

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21 kDa, possessed the highly conserved motif, ‘GCFWG’ in the N-terminus and ‘KGCNDPIRCY’ motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42 °C and pH 9.0 were optimum for NtMSRA4 activity. The K m and K cat values of NtMSRA4 were determined to be 40.04 μM and 0.048 S−1 in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H2O2 toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.

Although solution additives prevent protein misfolding, the mechanism remains elusive. In this paper, we compare the preventive effects of trans-1,2-cyclohexanediamine (1,2-CHDA) and trans-1,4-cyclohexanediamine (1,4-CHDA) on the heat-induced inactivation of ribonuclease A (RNase A) and lysozyme. These additives are more effective in preventing thermal inactivation of the proteins than guanidine (Gdn) and arginine (Arg). The results suggest two possibilities: (i) decrease in the hydrophobic interaction between unfolded protein molecules is indispensable for preventing protein association, and (ii) the electrostatic interaction between additives interacting with the hydrophobic residues of protein molecules plays an important role in preventing thermal inactivation of proteins.

The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6–9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca2+, Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.

Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn2+ and Mn2+ chelation solely and along with Ca2+. We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn2+ and Mn2+-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn2+ and Mn2+ treatment of CP after incubation with Ca2+ displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn2+ and Mn2+ treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca2+-treated CP, the tertiary structure integrity is intact upon Zn2+ and Mn2+ chelation. Interestingly, CP structural modifications upon Zn2+ and Mn2+ treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca2+-loaded state when treated with Zn2+ and Mn2+ ions. It can be suggested that Ca2+ not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.

Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in β-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that β-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced β-amyloid1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for β-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of β-amyloid1-42-induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with β-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis.

Reliable prediction of interface residues in transient complexes remains challenging, yet is highly desirable for the design of new drugs. The existing computational methods mainly rely on evolutionary information to identify these key residues, but evolutionary information may not be effective for the interface residues in all types of transient complexes, such as antigen–antibody complexes. Herein we combined B-factor with sequence profile and accessible surface area to predict these important residues using support vector machine (SVM). Furthermore, a post-processing method was developed to reduce the number of false positives recognized by SVM. The prediction results show that B-factor is an effective indicator for the interface residues in antigen–antibody complexes as well as those in other types of transient complexes. In addition, we found that the post-processing procedure made an important contribution to further improve the prediction performance. Consequently, the proposed approach could provide new insight into accurately predicting interface residues in different types of transient complexes.