Protein Polarimetry, Perfected: Specific Rotation Measurement for the Refracto-Polarimetric Detection of Cryptic Protein Denaturation

Amino acid composition and thermal stability of proteins

Transition temperature and thermal stability of proteins were studied in the presence and absence of boron. The observed midpoint of thermal denaturation (T m) of cytochrome c (Cyt c) at pH 9.2 was 68.8 °C, which in the presence of boron increased to 71.0 °C. For metmyoglobin, T m increased from 79.7 °C in the absence of boron to 83.5 °C in the presence of boron. Boron caused an increase of 10% in the reversibility of thermal denaturation of cytochrome c when compared with control. Activity measurements of the heat treated proteins and T m suggest an increased thermal stability toward inactivation and denaturation of heme proteins in the presence of boron.

Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. The I.lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean β-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both β-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I.lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I.lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). Enzymatic hydrolysis of soybean proteins by the I.lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Gum arabic from Acacia senegal (L) Willd. in Kenya

Role of weak interactions in thermal stability of proteins

MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

The three-dimensional structure of the histone-like HU protein from the mycoplasma Spiroplasma melliferum KC3 (HUSpm) was determined at 1.4 Å resolution, and the thermal stability of the protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HUSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HUSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HUSpm has a high denaturation temperature, comparable to that of HU proteins from thermophilic bacteria. To elucidate the structural basis of HUSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HUSpm revealed the motifs that play a key role in protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the protein, the absence of the internal cavity present at the dimer interface of some HU proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous proteins.

Comparison of polarimetry and crown ether-based HPLC chiral stationary phase method to determine (l)-amino acid optical purity

One hundred twelve human DNA sequences were analyzed with respect to dinucleotide frequency and amino acid composition. The variation in guanine and cytosine (G + C) content revealed: (1) at 2-3 and 3-1 doublet positions CG discrimination is attenuated at high G + C, but TA disfavor is enhanced, and (2) several amino acids are subject to G + C change. These findings have been reported in part for collections of sequences from various species. The present study confirms that in a single organism--the human--the G + C effects do exist. Aspects of the argument that connects G + C with protein thermal stability are also discussed.

An outdoor pot experiment was designed to study the changes of growth parameters, accumulation, and distribution of Cd in poplar (Populus nigra L.) during a prolonged exposure period (growing period of 17 months including three harvest points), allowing the consideration of time effects and prolonged adaptation to Cd stress. Simultaneously, changes to the antioxidant system in roots and leaves were monitored. It was demonstrated that poplar could adapt to the Cd-contaminated soils after prolonged exposure. Total Cd accumulation in the aerial parts of poplar, due to high biomass production and acceptable Cd accumulation parameters, implies that the tested poplar species could be a good candidate for Cd phytoextraction application as well as could be used as phytostabilizer of Cd in heavily polluted soil. Furthermore, the activity of the antioxidant machinery displays both a tissue- and exposure-specific response pattern to different Cd treatments, indicating that strict regulation of the antioxidant defense system is required for the adaptive response of poplar. In addition, this report highlights the importance of prolonged exposure studies of physiological responses of plants, especially for long-life-cycle woody species under heavy metal stress, since some misleading conclusions could be reached after shorter time periods.

The classification of natural gums. VI. Gum arabic derived from Acacia Senegal var. Kerensis from Kenya

The classification of natural gums. III. Acacia senegal and related species (gum arabic)