Potyviral Helper-Component Protease: Multifaced Functions and Interactions with Host Proteins.

The best-characterized functional motifs of the potyviral Helper-Component protease (HC-Pro) responding for aphid transmission, RNA silencing suppression, movement, symptom development, and replication are gathered in this review. The potential cellular protein targets of plant virus proteases remain largely unknown despite their multifunctionality. The HC-Pro catalytic domain, as a cysteine protease, autoproteolytically cleaves the potyviral polyproteins in the sequence motif YXVG/G and is not expected to act on host targets; however, 146 plant proteins in the clade containing this motif were searched in the UniProtKB database and are discussed.

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues.

Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation.

Non-target biotransformation enzymes as a target for triazole-zinc mixtures.

Triazoles inhibit lanosterol 14α-demethylase and block ergosterol biosynthesis in fungal pathogens. However, they also interact with other cytochrome P450 enzymes and influence non-target metabolic pathways. Disturbingly, triazoles may interact with essential elements.

Pythium oligandrum in plant protection and growth promotion: Secretion of hydrolytic enzymes, elicitors and tryptamine as auxin precursor.

Pythium is a genus of parasitic oomycetes which target plants and both nonvertebrate and vertebrate animals, including fish and mammalian species. However, several Pythium spp., such as P.

Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP).

Correction to "Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis".

[This corrects the article DOI: 10.1021/acsomega.1c00732.

Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis.

Fast photochemical oxidation of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of HO, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein's structure requires pinpointing of modifications using a bottom-up proteomic approach.

Motif orientation matters: Structural characterization of TEAD1 recognition of genomic DNA.

TEAD transcription factors regulate gene expression through interactions with DNA and other proteins. They are crucial for the development of eukaryotic organisms and to control the expression of genes involved mostly in cell proliferation and differentiation; however, their deregulation can lead to tumorigenesis. To study the interactions of TEAD1 with M-CAT motifs and their inverted versions, the K of each complex was determined, and H/D exchange, quantitative chemical cross-linking, molecular docking, and smFRET were utilized for structural characterization.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.

Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500).

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex.

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding.

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP.

The C-type lectin-like receptor Nkrp1b: Structural proteomics reveals features affecting protein conformation and interactions.

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared.

Impact of Chemical Cross-Linking on Protein Structure and Function.

Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far.

Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure.

Mouse Nkrp1a receptor is a C-type lectin-like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined.

Spectral density mapping at multiple magnetic fields suitable for (13)C NMR relaxation studies.

Standard spectral density mapping protocols, well suited for the analysis of (15)N relaxation rates, introduce significant systematic errors when applied to (13)C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested.

Probing the Ca(2+)-assisted π-π interaction during Ca(2+)-dependent protein folding.

Protein folding is governed by a balance of non-covalent interactions, of which cation-π and π-π play important roles. Theoretical calculations revealed a strong cooperativity between cation-π involving alkali and alkaline earth metal ions and π-π interactions, but however, no experimental evidence was provided in this regard. Here, we characterized a Ca(2+)-binding self-processing module (SPM), which mediates a highly-specific Ca(2+)-dependent autocatalytic processing of iron-regulated protein FrpC secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis.

Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR.

Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glucose allowing to produce homogenous soluble monomeric NKp30 in several formats needed for advanced NMR studies. Our optimized protocol now allows to produce routinely 10mg batches of these NKp30ex proteins per 1L of M9 production medium in four working days.

The combination of hydrogen/deuterium exchange or chemical cross-linking techniques with mass spectrometry: mapping of human 14-3-3ζ homodimer interface.

Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D.

Chemical cross-linking and H/D exchange for fast refinement of protein crystal structure.

A combination of chemical cross-linking and hydrogen-deuterium exchange coupled to high resolution mass spectrometry was used to describe structural differences of NKR-P1A receptor. The loop region extended from the compact core in the crystal structure was found to be closely attached to the protein core in solution. Our approach has potential to refine protein structures in solution within a few days and has very low sample consumption.

High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli.

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown.

Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight).

Investigation of thermal denaturation of barley nonspecific lipid transfer protein 1 (ns-LTP1b) by nuclear magnetic resonance and differential scanning calorimetry.

The process of thermal denaturation of a covalently modified form of barley grain nonspecific lipid transfer protein 1b (ns-LTP1b) was investigated by nuclear magnetic resonance (NMR) and differential scanning calorimetry up to 115 degrees C. The denaturation was found to be irreversible and highly cooperative. A method of numerical quantitative analysis allowing us to fit the NMR data to a transition state model without further simplification was developed.

NMR structure of the N-terminal domain of capsid protein from the mason-pfizer monkey virus.

The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six alpha-helix bundle and an N-terminal beta-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the beta-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV beta-hairpin in two mutants (i.