How anisotropic and isotropic atomic displacement parameters monitor protein covalent bonds rigidity: isotropic B-factors underestimate bond rigidity

Under the assumption that covalent bonds are rigid, it is possible to compare the estimations of rigidity based on anisotropic and isotropic B-factors. This is done by computing the difference of the mean-square displacements (Delta-u) of atoms A and Z along the covalent bond A–Z, which must be close to zero for a rigid bond. The analysis of a high-quality set of protein structures, refined at a resolution better than (or equal to) 0.8 Angstroms, showed that Delta-u is significantly close to zero when anisotropic B-factors are used, with an average 60% Delta-u reduction. This reduction is larger for larger B-factors and this suggests that care should be taken in data-mining procedures that involve isotropic B-factors, especially at lower resolution, when anisotropic B-factors cannot be determined and when the average B-factor increases.

Improvement of protein emulsion stability through glycosylated black bean protein covalent interaction with (−)-epigallocatechin-3-gallate

Stability improvement of emulsions stabilized by covalent conjugation with glycosylated black bean protein and EGCG (BBPI-G–EGCG) was studied through structure changes of proteins and emulsion properties.

Mass Spectrometry-Based Methodologies for Targeted and Untargeted Identification of Protein Covalent Adducts (Adductomics): Current Status and Challenges

Protein covalent adducts formed upon exposure to reactive (mainly electrophilic) chemicals may lead to the development of a wide range of deleterious health outcomes. Therefore, the identification of protein covalent adducts constitutes a huge opportunity for a better understanding of events underlying diseases and for the development of biomarkers which may constitute effective tools for disease diagnosis/prognosis, for the application of personalized medicine approaches and for accurately assessing human exposure to chemical toxicants. The currently available mass spectrometry (MS)-based methodologies, are clearly the most suitable for the analysis of protein covalent modifications, providing accuracy, sensitivity, unbiased identification of the modified residue and conjugates along with quantitative information. However, despite the huge technological advances in MS instrumentation and bioinformatics tools, the identification of low abundant protein covalent adducts is still challenging. This review is aimed at summarizing the MS-based methodologies currently used for the identification of protein covalent adducts and the strategies developed to overcome the analytical challenges, involving not only sample pre-treatment procedures but also distinct MS and data analysis approaches.

Spontaneous cross-linking of proteins at aspartate and asparagine residues is mediated via a succinimide intermediate

The breakdown of long-lived proteins (LLPs) is associated with aging, as well as disease; however, our understanding of the molecular processes involved is still limited. Of particular relevance, cross-linked proteins are often reported in aged tissues but the mechanisms for their formation are poorly understood. In the present study, sites of protein cross-linking in human ocular lenses were characterized using proteomic techniques. In long-lived lens proteins, several sites of cross-linking were found to involve the addition of Lys to Asp or Asn residues. Using model peptides containing Asp or Asn, a mechanism was elucidated that involves a succinimide intermediate. Succinimides formed readily from Asn at neutral pH, whereas a higher rate of formation from Asp peptides was observed at more acidic pHs. Succinimides were found to be relatively stable in the absence of nucleophiles. Since racemization of Asp residues, as well as deamidation of Asn, involves a succinimide intermediate, sites of d-Asp and isoAsp in LLPs should also be considered as potential sites of protein covalent cross-linking.

Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA

An efficient method to generate site-specific DNA–protein covalent complexes is described using intrastrand cross-linked DNA.