Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein–Protein Interaction

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural-diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity-directed synthesis (ADS), a function-driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small-molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.     

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.     

Protein structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

Protein side chains facilitate Mg/Al exchange in model protein binding sites.

Among the most frequent protein binding sites served by Mg(II), we identify those which have higher affinity towards Al(III). We also estimate the free energies of metal exchange for all these binding sites taking into account solvent effects explicitly. The obtained results show that thermodynamically favored Mg(II)/Al(III) exchange reactions take place at a number of these metal binding sites, which could possibly be related to some dysfunctions of Mg(II)-dependent biological processes. Additionally, they shed light on the molecular basis of the toxicity of Al(III) in living organisms.     

Protein open-access liquid chromatography/mass spectrometry

Each year increasing numbers of proteins are submitted for routine characterization by liquid chromatography/mass spectrometry (LC/MS). This paper reports a solution that transforms routine LC/MS analysis of proteins into a fully automated process that significantly reduces analyst intervention. The solution developed, protein open-access (OA) LC/MS, consists of web-enabled sample submission and registration, automated data processing, data interpretation, and report generation. Sample submissions and results are recorded in a LIMS that utilizes an Oracle database. The protein sequence is captured during the sample submission process, stored in the database, and utilized to determine the theoretical protein molecular weight. This calculated mass is used to set the parameters for transformation of the mass-to-charge spectra to the mass domain and evaluate the presence or absence of the desired protein. Three protein OA-LC/MS instruments have been deployed in our facility to support protein characterization, purification, and modification efforts.     

Protein identification based on matrix assisted laser desorption/ionization-post source decay-mass spectrometry.

Due to its very short analysis time, its high sensitivity and ease of automation, matrix-assisted laser desorption/ionization (MALDI)-peptide mass fingerprinting has become the preferred method for identifying proteins of which the sequences are available in databases. However, many protein samples cannot be unambiguously identified by exclusively using their peptide mass fingerprints (e.g., protein mixtures, heavily posttranslationally modified proteins and small proteins). In these cases, additional sequence information is needed and one of the obvious choices when working with MALDI-mass spectrometry (MS) is to choose for post source decay (PSD) analysis on selected peptides. This can be performed on the same sample which is used for peptide mass fingerprinting. Although in this type of peptide analysis, fragmentation yields are very low and PSD spectra are often very difficult to interpret manually, we here report upon our five years of experience with the use of PSD spectra for protein identification in sequence (protein or expressed sequence tag (EST)) databases. The combination of peptide mass fingerprinting and PSD and analysis described here generally leads to unambiguous protein identification in the amount of material range generally encountered in most proteome studies.     

离子液与蛋白质和核酸相互作用的研究

离子液因其具有良好的生物兼容性和独特的理化性质,近年来在生物催化和生物大分子蛋白质与核酸的分离分析领域得到广泛应用。离子液与生物大分子相互作用的研究是离子液相关理论与应用研究的基础,有关离子液与蛋白质和核酸相互作用的机理研究受到关注。本文简要介绍了常用离子液的分类,离子液与蛋白质分子作用的机理,离子液与核酸分子作用的机理,以及离子液在酶催化反应、生物分子分离、生物分子电化学分析和毛细管电泳分析中的应用,并主要综述了近年的相关研究和应用进展。     

Protein folding at emulsion oil/water interfaces

It has long been known that proteins change their conformation upon adsorption to emulsion oil/water interfaces. However, it is only recently that details of the specifics of these structural changes have emerged. The development of synchrotron radiation circular dichroism (SRCD), combined with advances in FTIR spectroscopy, has allowed the secondary and tertiary structure of proteins adsorbed at emulsion oil/water interfaces to be studied. SRCD in particular has provided quantitative information and has enabled new insights into the mechanisms and forces driving protein structure re-arrangement to be achieved.The extent of conformational re-arrangement of proteins at emulsion interfaces is influenced by several factors including; the inherit flexibility of the protein, the distribution of hydrophobic/hydrophilic domains within the protein sequence and the hydrophobicity of the oil phase. In general, proteins lose much of their tertiary structure upon adsorption to the oil/water interface and have considerable amounts of non-native secondary structure. Two key conformations have been identified in the structure of proteins at interfaces, intermolecular β-sheet and α-helix. The preferred conformation appears to be the α-helix which is the most compact amphipathic conformation at the oil/water interface. The polarity of the oil phase can have a considerable influence on the degree of protein conformational re-arrangement because it acts as a solvent for hydrophobic amino acids. The new conformation of proteins at interfaces also means that proteins undergo less heat induced re-arrangement at interfaces than in solution. Different conformations of proteins at interfaces impact on emulsification capability, emulsion stability and protein/emulsion digestion. Hence advances in the understanding of protein conformation at interfaces can help to identify suitable proteins and conditions for the preparation of emulsion based food products.