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The Biochemical Journal Nov 2018Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are vital eukaryotic signaling elements that convey information from ligand-regulated G protein-coupled... (Review)
Review
Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are vital eukaryotic signaling elements that convey information from ligand-regulated G protein-coupled receptors (GPCRs) to cellular effectors. Heterotrimeric G protein-based signaling pathways are fundamental to human health [ (2007) , 994-1005] and are the target of >30% of pharmaceuticals in clinical use [ (2013) , 1676-1694; (2017) , 829-842]. This review focuses on phosphorylation of G protein subunits as a regulatory mechanism in mammals, budding yeast, and plants. This is a re-emerging field, as evidence for phosphoregulation of mammalian G protein subunits from biochemical studies in the early 1990s can now be complemented with contemporary phosphoproteomics and genetic approaches applied to a diversity of model systems. In addition, new evidence implicates a family of plant kinases, the receptor-like kinases, which are monophyletic with the interleukin-1 receptor-associated kinase/Pelle kinases of metazoans, as possible GPCRs that signal via subunit phosphorylation. We describe early and modern observations on G protein subunit phosphorylation and its functional consequences in these three classes of organisms, and suggest future research directions.
Topics: Animals; Heterotrimeric GTP-Binding Proteins; Humans; Mammals; Phosphorylation; Plants; Protein Binding; Protein Subunits; Saccharomyces cerevisiae; Signal Transduction
PubMed: 30413679
DOI: 10.1042/BCJ20160819 -
Methods in Molecular Biology (Clifton,... 2021Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of...
Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of expression system, detergents, and purification tags is therefore an important decision. Here, we present a protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein complexes for structural and functional studies.
Topics: Chromatography, Affinity; Chromatography, Gel; Escherichia coli; Gene Expression; Membrane Proteins; Multiprotein Complexes; Plasmids; Promoter Regions, Genetic; Protein Multimerization; Protein Subunits; Recombinant Proteins
PubMed: 33301109
DOI: 10.1007/978-1-0716-1126-5_1 -
Cell Calcium 2007For a long time the auxiliary beta-subunit of voltage-gated calcium channels was thought to be engaged exclusively in the regulation of calcium channel function,... (Review)
Review
For a long time the auxiliary beta-subunit of voltage-gated calcium channels was thought to be engaged exclusively in the regulation of calcium channel function, including gating, intracellular trafficking, assembly and membrane expression. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins (MAGUK) that comprises a series of protein interaction motifs. Two such domains, a Src homology 3 and a guanylate kinase domain are present in the beta-subunit. Recently, it was shown that this subunit interacts with a variety of proteins and regulates diverse cellular processes ranging from gene expression to hormone secretion and endocytosis. In light of these new findings, the beta-subunit deserves to be promoted to the category of multifunctional regulatory protein. Some of these new functions support a tighter regulation of calcium influx through voltage-gated calcium channels and others apparently serve channel unrelated processes. Here we discuss a variety of protein-protein interactions held by the beta-subunit of voltage-gated calcium channels and their functional consequences. Certainly the identification of additional binding partners and effector pathways will help to understand how the different beta-subunit-mediated processes are interwoven.
Topics: Animals; Calcium; Calcium Channels; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Dynamins; Monomeric GTP-Binding Proteins; Protein Subunits
PubMed: 17629941
DOI: 10.1016/j.ceca.2007.05.009 -
Proteomics Aug 2015Although the number of protein-encoding genes in the human genome is only about 20 000 not far from the amount found in the nematode worm genome, the number of proteins... (Review)
Review
Although the number of protein-encoding genes in the human genome is only about 20 000 not far from the amount found in the nematode worm genome, the number of proteins that are translated from these sequences is larger by several orders of magnitude. A number of mechanisms have evolved to enable this diversity. For example, genes can be alternatively spliced to create multiple transcripts; they may also be translated from different alternative initiation sites. After translation, hundreds of chemical modifications can be introduced in proteins, altering their chemical properties, folding, stability, and activity. The complexity is then further enhanced by the various combinations that are generated from the assembly of different subunit variants into protein complexes. This, in turn, confers structural and functional flexibility, and endows the cell with the ability to adapt to various environmental conditions. Therefore, exposing the variability of protein complexes is an important step toward understanding their biological functions. Revealing this enormous diversity, however, is not a simple task. In this review, we will focus on the array of MS-based strategies that are capable of performing this mission. We will also discuss the challenges that lie ahead, and the future directions toward which the field might be heading.
Topics: Computational Biology; Mass Spectrometry; Models, Molecular; Protein Conformation; Protein Processing, Post-Translational; Protein Subunits
PubMed: 25727951
DOI: 10.1002/pmic.201400517 -
Bioorganic Chemistry Mar 2023The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for...
The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.
Topics: Alkenes; Protein Subunits; Heterocyclic Compounds; Amino Acids; Cycloaddition Reaction
PubMed: 36642019
DOI: 10.1016/j.bioorg.2023.106359 -
Thermokinetic Analysis of Protein Subunit Exchange by Variable-Temperature Native Mass Spectrometry.Biochemistry Dec 2019Many protein complexes are assembled from a varying number of subunits, which are continuously exchanging with diverse time scales. This structural dynamics is...
Many protein complexes are assembled from a varying number of subunits, which are continuously exchanging with diverse time scales. This structural dynamics is considered to be important for many regulatory and sensory adaptation processes that occur . We have developed an accurate method for monitoring protein subunit exchange by using native electrospray ionization mass spectrometry (ESI-MS), exemplified here for an extremely stable Rad50 zinc hook (Hk) dimer assembly, Zn(Hk). The method has two steps: appropriate protein/peptide mutation and native ESI-MS analysis using a variable-temperature sample inlet. In this work, two Hk mutants were produced, mixed with wild-type Hk, and measured at three different temperatures. A thermokinetic analysis of heterodimer formation allowed us to determine the enthalpy, entropy, and Gibbs free energy of activation for subunit exchange, showing that the reaction is slow and associated with a high enthalpic barrier, consistent with the exceptionally high stability of the Zn(Hk) assembly.
Topics: Amino Acid Sequence; DNA-Binding Proteins; Kinetics; Mass Spectrometry; Models, Molecular; Protein Conformation; Protein Subunits; Temperature
PubMed: 31790206
DOI: 10.1021/acs.biochem.9b00911 -
Chembiochem : a European Journal of... Sep 2002
Review
Topics: Animals; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Humans; Protein Subunits; Sex Attractants; Transducin; Yeasts
PubMed: 12210980
DOI: 10.1002/1439-7633(20020902)3:9<815::AID-CBIC815>3.0.CO;2-E -
Food Chemistry Dec 2023Prolyl endopeptidase can partially degrade soybean protein B subunit and alleviate soy sauce secondary precipitate. In this study, the influences of ultrasound-assisted...
Prolyl endopeptidase can partially degrade soybean protein B subunit and alleviate soy sauce secondary precipitate. In this study, the influences of ultrasound-assisted prolyl endopeptidase on the degradation of soybean protein B subunit of soy sauce and primary mechanism were investigated using SDS-PAGE, MALDI-TOF-MS, circular dichromatic spectrometer, fluorescence spectra, etc. Results showed that ultrasound-assisted prolyl endopeptidase enhanced 72% degradation rate of B subunit and reduced soy sauce secondary precipitate remarkably, meanwhile significantly increased content of organic taste compounds of soy sauce compared with control (p < 0.05). Sonication markedly reduced percentage of α-helix and increased percentage of random coil, made hydrophobic amino acids inside prolyl endopeptidase exposed to its surface and enhanced its flexibility, which facilitated the binding of prolyl endopeptidase active center with B subunit and finally enhanced the latter's degradation rate and appearance quality of soy sauce. This work laid a foundation for solving soy sauce secondary precipitate.
Topics: Soy Foods; Soybean Proteins; Prolyl Oligopeptidases; Molecular Weight; Protein Subunits; Fermentation; Protein Structure, Secondary; Sonication
PubMed: 37506662
DOI: 10.1016/j.foodchem.2023.136972 -
Methods (San Diego, Calif.) Mar 2013Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge...
Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes.
Topics: COP9 Signalosome Complex; Chemical Fractionation; Humans; Mass Spectrometry; Multiprotein Complexes; Peptide Hydrolases; Protein Denaturation; Protein Subunits; Proteomics
PubMed: 23296018
DOI: 10.1016/j.ymeth.2012.12.013 -
Biomolecules Nov 2020The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research,... (Review)
Review
The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, mutants lacking (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.
Topics: Bacterial Proteins; DNA-Directed RNA Polymerases; Mutant Proteins; Protein Subunits; Structure-Activity Relationship; Transcription Factors
PubMed: 33238579
DOI: 10.3390/biom10111588