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BBA - Proteins and Proteomics (v.1814, #9)

Editorial Board (pp. i).

Characterization of the single-stranded DNA binding protein pV^VGJΦ of VGJΦ phage from Vibrio cholerae by Alina Falero; Andy Caballero; Sonia Trigueros; Perez Celso Pérez; Javier Campos; Karen Marrero; Rafael Fando (pp. 1107-1112).

pV^VGJΦ, a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC–SEC. Moreover, isoelectrofocusing showed an isoelectric point for pV^VGJΦ of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pV^VGJΦ binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at −20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pV^VGJΦ protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pV^VGJΦ interesting for biotechnological applications.► pV^VGJΦ a DNA binding protein of phage VGJΦ has a general affinity for ssDNA. ► SEC revealed a major species of 27.0kDa, suggesting a homodimeric protein. ► pV^VGJΦ binds ssDNA at extreme temperatures, stable after extended times. ► After a year at –20°C the protein kept its biological activity and oligomericity. ► pV^VGJΦ is interesting and attractive for further application in biotechnology.

Keywords: VGJΦ phage; pV; ^VGJΦ; protein; DNA binding protein; Thermostability

Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates by Emily J. Fogle; Michael D. Toney (pp. 1113-1119).

Diaminopimelate decarboxylase (DAPDC) and ornithine decarboxylase (ODC) are pyridoxal 5'-phosphate dependent enzymes that are critical to microbial growth and pathogenicity. The latter is the target of drugs that cure African sleeping sickness, while the former is an attractive target for antibacterials. These two enzymes share the (β/α)₈ (i.e., TIM barrel) fold with alanine racemase, another pyridoxal 5'-phosphate dependent enzyme critical to bacterial survival. The active site structural homology between DAPDC and ODC is striking even though DAPDC catalyzes the decarboxylation of a D stereocenter with inversion of configuration and ODC catalyzes the decarboxylation of an L stereocenter with retention of configuration. Here, the structural and mechanistic bases of these interesting properties are explored using reactions of alternate substrates with both enzymes. It is concluded that simple binding determinants do not control the observed stereochemical specificities for decarboxylation, and a concerted decarboxylation/proton transfer at Cα of the D stereocenter of diaminopimelate is a possible mechanism for the observed specificity with DAPDC.► The stereospecificity of DAPDC and ODC has been analyzed using alternate substrates. ► Simple binding determinants do not control reactivity. ► A concerted transition state is proposed for DAPDC to explain its stereospecificity.

Keywords: Pyridoxal 5'phosphate; Decarboxylase; Decarboxylation; TIM barrel; Reaction specificity; Enzyme mechanisms

Structural stability of Staphylococcus xylosus lipase is modulated by Zn²⁺ ions by Jean Borges Bertoldo; Guilherme Razzera; Javier Vernal; Fábio Cristiano Angonesi Brod; Ana Carolina Maisonnave Arisi; Hernán Terenzi (pp. 1120-1126).

Lipases are well-known enzymes extensively used in industrial biotransformation processes. Besides, their structural and catalytic characteristics have attracted increasing attention of several industries in the last years. In this work, we used biophysical and molecular modeling tools to assess structural properties of Staphylococcus xylosus lipase (SXL). We studied the thermal unfolding of this protein and its zinc-dependent thermotolerance. We demonstrated that SXL is able to be active and stable at moderate temperatures, but this feature is only acquired in the presence of Zn²⁺. Such characteristic indicates SXL as a zinc-dependent metallolipase.► Biophysical and molecular modeling tools to assess structural properties of SXL lipase. ► Thermal unfolding and its zinc-dependent thermotolerance and activity were studied. ► SXL is active and stable at moderate temperatures, this feature is acquired in the presence of Zn²⁺.

Keywords: Metallolipase; Circular dichroism; Thermal stability; Thermostable lipase

Characterization of calcium ion sensitive region for β-Mannanase from Streptomyces thermolilacinus by Yuya Kumagai; Hirokazu Usuki; Yukihiro Yamamoto; Akihiro Yamasato; Jiro Arima; Takafumi Mukaihara; Tadashi Hatanaka (pp. 1127-1133).

Despite the widespread industrial applications of β-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on β-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding β-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7–9°C high temperature in the presence of Ca²⁺ compared with that in the absence of Ca²⁺. These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca²⁺. StMan, but not TfMan, required Ca²⁺ for the hydrolysis activity. To identify the Ca²⁺ sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca²⁺ and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca²⁺ sensitive site.► Ca²⁺ sensitive regions of β-mannanase are investigated by using chimeras between StMan and TfMan. ► Thermal stabilities of StMan and TfMan are enhanced by Ca²⁺. ► The hydrolysis activity of StMan is enhanced by Ca²⁺.

Keywords: β-Mannanase; Streptomyces; Thermobifida; Calcium ion dependent activity; Chimera; Thermal stability

Identification of the phosphorylation sites in the survival motor neuron protein by protein kinase A by Chia-Yen Wu; Amelia Curtis; Yong Seok Choi; Miho Maeda; Mary Jue Xu; Amanda Berg; Upasana Joneja; Robert W. Mason; Kelvin H. Lee; Wenlan Wang (pp. 1134-1139).

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.► SMN can be phosphorylated by protein kinase A in vivo and in vitro. ► SMN is phosphorylated at five sites by protein kinase A. ► Mutation of protein kinase A sites affects association of SMN with Gemin2 and Gemin8.

Keywords: Spinal muscular atrophy; Survival motor neuron protein; Protein kinase A; Phosphorylation site

Design and characteristics of a stable protein scaffold for specific binding based on variable lymphocyte receptor sequences by Wezner-Ptasinska Magdalena Wezner-Ptasińska; Daniel Krowarsch; Jacek Otlewski (pp. 1140-1145).

Variable lymphocyte receptors (VLRs) serve as antigen binding proteins in jawless vertebrates. Their relatively low molecular weight makes VLRs an interesting alternative to antibodies in biotechnological applications. A typical VLR comprises several unique motifs called leucine-rich repeats (LRRs). Using consensus approach we designed a novel VLR protein (called dVLR) containing six LRR repeats based on a sea lamprey receptor sequence. The designed protein was expressed in Escherichia coli in a soluble, native form and showed very favorable biophysical properties. Recombinant dVLR is monomeric in solution and preserves its secondary structure within the pH range 3.0 to 11.0 and tertiary structure between pH 4.0 and 10.0. It undergoes reversible thermal denaturation in a broad pH range (4.0 to 10.0). The maximal denaturation temperature of 73.9°C is observed at pH 6.0, 0.3M NaCl. Chemical denaturation of dVLR at pH 7.5 is a cooperative two-state process with a midpoint at 3.3M GdmCl and a very high free energy change of unfolding in the absence of denaturant equal to 14.1kcal/mol. The biophysical properties of dVLR make it highly suitable for biotechnological applications such as generation of specific ligand-binding molecules.► Design of novel variable lymphocyte receptor protein (dVLR). ► dVLR remains monomeric in various protein concentration. ► dVLR shows high thermostability over broad pH range. ► The highest stability was observed at pH 6.0 with denaturation temperature 73.9 °C. ► dVLR is suitable for biotechnological applications.

Keywords: Abbreviations; VLR; variable lymphocyte receptor; VLRB; variable lymphocyte receptor type B; dVLR; designed VLR; LRR; leucine-rich repeat; LRRNT; leucine-rich repeat N-terminal capping region; LRR1; the first leucine-rich repeat; LRRV; leucine-rich repeat variable; LRRVe; leucine-rich repeat variable end; LRRCP; leucine-rich repeat connecting peptide; LRRCT; leucine-rich repeat C-terminal capping region; CD; circular dichroism; FL; fluorescence spectroscopy; ΔG; _den; H; ₂; ⁰; free energy change of unfolding; GdmCl; guanidinium chloride; TEV; tobacco etch virusVariable lymphocyte receptor; Leucine-rich repeat; Consensus sequence design; Protein stability

Effects of monopropanediamino-β-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD by Marylène Vandevenne; Gilles Gaspard; El Mustapha Belgsir; Manilduth Ramnath; Yves Cenatiempo; Daniel Marechal; Mireille Dumoulin; Jean-Marie Frere; André Matagne; Moreno Galleni; Patrice Filee (pp. 1146-1153).

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. β-Cyclodextrin (β-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified β-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A β-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of β-CD derivatives on the stability of proteins is discussed.► We studied the effects of a cyclodextrin on the stability of a hybrid protein. ► This cyclodextrin showed anti-aggregating properties during thermal denaturation. ► Addition of the cyclodextrin also increased the protein stability over the time. ► We suggested a model to explain the stabilizing properties of the cyclodextrin.

Keywords: Abbreviations; ChBD; chitin binding domain of the human macrophage chitotriosidase; BCD07056; monopropanediamino-β-cyclodextrin; PEG; polyethylene glycol; Far UV-CD; circular dichroism in far ultraviolet; β-CD; β-cyclodextrin; T; _M; temperature of mid-transition; ANS; 1-Anilino-8-naphthalenesulfonateProtein aggregation; Cyclodextrins; Protein folding; Hybrid protein; Cyclodextrin-protein; Interaction

Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: The carbohydrate binding mechanism involves residual sialidase activity by Pablo Oppezzo; Gonzalo Obal; Martín A. Baraibar; Otto Pritsch; Pedro M. Alzari; Alejandro Buschiazzo (pp. 1154-1161).

Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite–host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.► First crystal structure of transialidase-like lectin iTS in complex with carbohydrate. ► The sialyl moiety is not visible in density, while lactose remains bound. ► iTS is confirmed to retain a residual sialidase activity by enzyme kinetics assays. ► iTS is able to bind sialylated glycoproteins as measured by surface plasmon resonance. ► The molecular details explain the lectin behavior of this trypanosomal protein family.

Keywords: Abbreviations; TS; trans-sialidase; iTS; inactive trans-sialidase-like lectin; rmsd; root mean squared deviation; 4-MUNANA; 4-methylumbelliferyl-N-acetyl-neuraminic acid; SPR; surface plasmon resonance; wt; wild-type; RU; resonance unitLectin; Trypanosoma; Sialic acid; Enzyme; Glycobiology; Protein crystallography

Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum by André O. Hudson; Ayelet Klartag; Charles Gilvarg; Renwick C.J. Dobson; Felipe Garbelini Marques; Thomas Leustek (pp. 1162-1168).

Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The B. fragilis Ddh ortholog shows low homology with other examples of Ddh and this species belongs to a phylum, the Bacteriodetes, not previously known to contain this enzyme. By contrast, the C. thermocellum ortholog is well conserved with known examples of Ddh. Using in vitro and in vivo assays both the B. fragilis and C. thermocellum enzymes were found to be authentic examples of Ddh, displaying kinetic properties typical of this enzyme. The result indicates that B. fragilis contains a sequence diverged form of Ddh. Phylogenomic analysis of the microbial genome database revealed that 77% of species with a Ddh ortholog also contain a second pathway for DAP biosynthesis suggesting that Ddh evolved as an ancillary mechanism for DAP biosynthesis.► Locus tags Bf3481 and Cthe_0922 encode authentic forms of diaminopimelate dehydrogenase. ► Bf3481 defines a previously uncharacterized diaminopimelate dehydrogenase (Ddh). ► Ddh frequently occurs in the same genome as another diaminopimelate pathway. ► Ddh has a restricted phylogenomic distribution. ► Hypothesis: Ddh evolved recently as an ancillary diaminopimelate biosynthesis mechanism.

Keywords: Abbreviations; THDPA; tetrahydrodicolinate; DAP; diaminopimelate; m; - and; L,L; -isomers; Ddh; m; -DAP dehydrogenase; DapL; L,L; -diaminopimelate aminotransferase; DapD; THDPA acyltransferaseLysine; Diaminipimelate; Dehydrogenase; Phylogenomics

Local/bulk determinants of conformational stability of exchangeable apolipoproteins by Alexander D. Dergunov (pp. 1169-1177).

GuHCl-induced denaturation of human plasma apoA-I, apoA-II, apoA-IV, apoE3 and three recombinant apoE isoforms in solution and discoidal complexes with phosphatidylcholine (only plasma proteins) was studied. The protein conformational stability (Δ G(H₂O)) and a slope of linear dependence of free energy of unfolding on GuHCl concentration ( m-value) were estimated with the three equilibrium schemes. The data for all proteins, except apoA-II, fit with the three-state model, thus evidencing two-domain structure. The predicted folding rate of the four apoE in solution correlated with conformational stability. The dependence disappeared at the inclusion of apoA-I and apoA-IV into analysis and the m-values, adjusted for residue number in helices ( m_rh), differed between those for apoE and apoA-I/apoA-IV. However, the m_rh-values for six proteins correlated positively with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The difference between the adjusted Δ G_rh(H₂O) values for apolipoproteins in complexes and in solution decreased at the increase of reduced temperature ( T_obs− T_t)/ T_t. The induction of intrinsic disorder by arginine residues may be of primary importance in metabolism and function of exchangeable apolipoproteins, while their stability in nascent discoidal HDL is controlled by the physical state of phosphatidylcholine.► Arginine induces intrinsic disorder in proteins associated with cardiovascular events. ► Apolipoproteins in nascent discoidal HDL possess the unifying two-domain structure. ► Dynamics of HDL lipid matrix determines apolipoprotein internal flexibility.

Keywords: Apolipoprotein folding; Domain structure; Stability; m; -value; Lipid–protein interactions

Mobilization of storage proteins in soybean seed ( Glycine max L.) during germination and seedling growth by Hyun Tae Kim; Ung-Kyu Choi; Hyun Su Ryu; Su Jin Lee; Oh-Shin Kwon (pp. 1178-1187).

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds ( Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.► Proteomic analysis showed temporal profiles of protein expression during soybean seed germination. ► Major proteins identified were subunits of glycinin and β-conglycinin. ► The acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. ► The degradation pattern of β-conglycinin appeared to be similar to that of glycinin but at a faster rate. ► The mobilization of their subunits is differentially regulated for seed germination and seedling growth.

Keywords: Proteomic; Germination; Glycine max; Glycinin; β-conglycinin

Dynamics of heme in hemoproteins: proton NMR study of myoglobin reconstituted with iron 3-ethyl-2-methylporphyrin by Sandrine Juillard; Soizic Chevance; Arnaud Bondon; Gérard Simonneaux (pp. 1188-1194).

The asymmetric 3-ethyl-2-methylporphyrin iron complex was synthetized and inserted into apomyoglobin. UV–visible spectroscopic studies demonstrated the capacity of iron to coordinate different exogenous axial ligands in ferrous and ferric forms. The position of synthetic heme into the hydrophobic pocket of the reconstituted myoglobin was investigated by^¹H NMR spectroscopy. In absence of exogenous ligand, signals of the synthetic prosthetic group were not detected, suggesting a rotational disorder of the synthetic porphyrin into the heme pocket. This direct interconversion behavior is favored since site-specific interactions between the poorly substituted heme and protein in the chiral hydrophobic cavity were weak. Complexion of cyanide to the iron allowed to quench partially the heme reorientation and two interconvertible forms, around the meso- Cα– Cγ axis, were detected in solution.► Asymmetric 3-ethyl-2-methylporphyrin iron complex was inserted into apomyoglobin. ► Cyanide complexation to iron allowed to quench partially the heme reorientation. ► Two interconvertible forms were detected in solution by NMR.

Keywords: Nuclear magnetic resonance; Reconstituted myoglobin; Iron porphyrin; Proton assignment; Hemin

Aminopropyl-functionalized cubic Ia3d mesoporous silica nanoparticle as an efficient support for immobilization of superoxide dismutase by Mojtaba Falahati; Leila Ma'mani; Ali Akbar Saboury; Abbas Shafiee; Alireza Foroumadi; Ali Reza Badiei (pp. 1195-1202).

In this research, the immobilization of superoxide dismutase (SOD) onto aminopropyl-functionalized KIT-6 [n-PrNH₂-KIT-6] was investigated. This organo-functionalized mesoporous silica nanoparticle was prepared using a non-ionic surfactant and was fully characterized by XRD, nitrogen adsorption–desorption isotherm assay, IR and TGA techniques. An activity assay demonstrated that the immobilized SOD had a higher activity than the free enzyme. Further investigations using FT-IR, circular dichroism (CD), and probe 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity measurements indicated that the structure of the enzyme did not change upon binding to the mesoporous silica, and that immobilized SOD was also less affected by higher temperatures. The melting temperatures of the free and immobilized enzymes were measured by differential scanning calorimetry (DSC), which showed that a fraction of immobilized enzyme was more stable and revealed that immobilized enzyme was partly reversible.► Immobilization of SOD. ► [ n-PrNH2-KIT-6]. ► More active and stable SOD.

Keywords: Mesoporous silica; KIT-6; Superoxide dismutase; Immobilization; Functionalization

In vitro interaction between the ammonium transport protein AmtB and partially uridylylated forms of the P_II protein GlnZ by Thiago E. Rodrigues; Victor E.P. Souza; Rose A. Monteiro; Edileusa C.M. Gerhardt; Araujo Luíza M. Araújo; Leda S. Chubatsu; Emanuel M. Souza; Fábio O. Pedrosa; Luciano F. Huergo (pp. 1203-1209).

The ammonium transport family Amt/Rh comprises ubiquitous integral membrane proteins that facilitate ammonium movement across biological membranes. Besides their role in transport, Amt proteins also play a role in sensing the levels of ammonium in the environment, a process that depends on complex formation with cytosolic proteins of the P_II family. Trimeric P_II proteins from a variety of organisms undergo a cycle of reversible posttranslational modification according to the prevailing nitrogen supply. In proteobacteria, P_II proteins are subjected to reversible uridylylation of each monomer. In this study we used the purified proteins from Azospirillum brasilense to analyze the effect of P_II uridylylation on the protein's ability to engage complex formation with AmtB in vitro. Our results show that partially uridylylated P_II trimers can interact with AmtB in vitro, the implication of this finding in the regulation of nitrogen metabolism is discussed. We also report an improved expression and purification protocol for the A. brasilense AmtB protein that might be applicable to AmtB proteins from other organisms.► In this study we examined how P_II protein uridylylation affects interaction with AmtB. ► We observed that fully uridylylated P_II cannot interact with AmtB. ► We conclude that partially uridylylated P_II interacts with AmtB.

Keywords: Azospirillum brasilense; Nitrogen metabolism; AmtB; P; _II; protein

Direct-reversible binding of small molecules to G protein βγ subunits by A.M.P.B. Seneviratne; Michael Burroughs; Ernest Giralt; Alan V. Smrcka (pp. 1210-1218).

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, β, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gβγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gβγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gβγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the k_off was slow for most of the compounds in physiological buffers, they could be removed from Gβγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gβγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gβγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gβγ. This is highly relevant to development of Gβγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.► An SPR method is presented for examining small molecule binding to G protein βγ subunits. ► SPR analysis demonstrates direct reversible binding of small molecules to Gβγ. ► Rate constants for small molecule binding and dissociation are surprisingly slow. ► Mass spectrometry demonstrates a predominantly 1:1, non-covalent binding mode for gallein. ► Radioligand binding supports a 1:1 binding mode of gallein to Gβγ.

Keywords: Abbreviations; GPCR; G protein-coupled receptor; SIGK; peptide with sequence SIGKAFKILGYPDYD; Gβγ; Heterotrimeric G protein βγ subunits; DTT; Dithiothreitol; SPR; Surface Plasmon Resonance; ELISA; Enzyme Linked Immunosorbent Assay; PLC; Phospholipase C; GRK2; G protein-coupled receptor kinase 2; TBS; Tris-buffered SalineG protein; Surface plasmon resonance; Protein–ligand interactions; Non-covalent binding

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BBA - Proteins and Proteomics (v.1814, #9)