Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers

A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand. The ATP, installed with this cross-linker at the γ-position, successfully transferred a coumarin onto ATP-binding proteins using only UV-irradiation.     

Enzyme occupancy measurement of intracellular protein tyrosine phosphatase 1B using photoaffinity probes

Protein tyrosine phosphatase 1B (PTP1B) is believed to be one of the enzymes involved in down-regulating the insulin receptor and is a drug target for the treatment of type II diabetes. To better understand the in vitro and in vivo behavior of PTP1B inhibitors, a cell-based assay to directly measure enzyme occupancy of PTP1B by inhibitors using photoaffinity labeling was developed. Two photoaffinity probes were synthesized containing the photolabile diazirine moiety. These photoprobes were specific for PTP1B and T-cell protein tyrosine phosphatase over CD45, with the most potent photoprobe having an IC(50) value of 0.2nM for PTP1B. Activation of the photoprobes with a 40-W UV lamp in the presence of purified AspTyrLysAspAspAspAspLys (Flag)-PTP1B formed a 1:1 irreversible adduct with the enzyme. The photolabeling was competed by known PTP1B inhibitors, vanadate, and the peptide inhibitor N-benzoyl-l-glutamyl-[4-phosphono(difluoromethyl)]-l-phenylalanyl-[4-phosphono(difluoromethyl)]l-phenylalanineamide (BzN-EJJ-amide). In HepG2 (human hepatoma cell line) cells, endogenous PTP1B was labeled by the UV-activated photoprobes in both lysed and intact cells. Enzyme occupancy measurements were conducted with a series of PTP1B inhibitors using the photoprobe affinity assay. Several compounds were shown to bind to endogenous PTP1B in the HepG2 intact cells.     

Diazirine-containing tag-free RNA probes for efficient RISC-loading and photoaffinity labeling of microRNA targets

We designed and synthesized a photo-reactive and tag-free RNA probe for the identification of microRNA (miRNA) targets. To synthesize the RNA probe, we designed a novel nucleoside analog 1-O-[3-ethynyl-5-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-β-d-ribofuranose containing aryl trifluoromethyl diazirine and ethynyl moieties. The RNA probe containing this analog was observed to form crosslinks with complementary RNA by UV irradiation and was rapidly tagged by Cu-catalyzed azide alkyne cycloaddition (CuAAC). In addition, the tag-free and photo-reactive miRNA-145 probe showed comparable gene silencing activity to that of unmodified miRNA-145. Therefore, miRNA probes containing the nucleoside analog are promising candidates for the identification of target mRNAs of miRNAs.     

特异性的蛋白小分子荧光探针及其标记技术

活体蛋白荧光标记技术已经被广泛应用于蛋白质功能的可视化研究中。荧光蛋白常被用来研究蛋白质在生物体内的表达和定位,但由于它本身体积比较大,往往会影响目标蛋白的生物活性。特异性的小分子荧光探针以其体积小、膜透性好、背景噪音低以及制备方便的优点成为蛋白质研究的一个有力工具。本文将简要介绍近几年来各类特异性小分子蛋白荧光探针的研究进展。     

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent D-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K(i) values of 22+/-5 microM for TIPDG and 12+/-2 microM for BzG. These K(i) values are comparable to those of their parent compounds arbutin (25+/-6 microM) and phlorizin (8+/-1 microM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure-function relationship of SGLT1 and other SGLT1 family transporter proteins.     

A method for site‐specific labeling of multiple protein thiols

We present a generic method for the site‐specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is removed, making available a reactive dithiol site for labeling with a second probe. For proof‐of‐principle, single and triple Cys mutants of the sulphate binding protein of an ABC transporter were constructed. The closely spaced thiols were engineered on the basis of the crystal structure of the protein and placed in different types of secondary structure elements and at different spacing. We show that phenyl arsenic oxide is a good protecting group for thiols spaced 6.3–7.3 Å. Proteins were labeled with two different fluorescent labels and the labeling ratios were determined with UV‐Vis spectroscopy and MALDI‐Tof mass spectrometry. The average labeling efficiency was ～80% for the single thiol and 65–90% for the dithiol site.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc₁ complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc₁ complex were measured. Mixtures of bc₁ complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dualwavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 13 derivative produced a diminution of the polypeptide of the bc₁ complex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c₁, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc₁ complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.     

Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A new property of twitchin to restrict the “rolling” of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin-myosin interaction by “freezing” tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.     

Interaction of the chloroplast ATP synthetase with the photoreactive nucleotide 3′-O-(4-benzoyl)benzoyl adenosine 5''-diphosphate

The photoreactive nucleotide 3'-O-(4-benzoyl)benzoyl ADP (BzADP) is not a substrate for photophosphorylation but is a strong competitive inhibitor (K_i 2-25μM) with respect to ADP and ATP in photophosphorylation or ATP hydrolysis and P_i-ATP exchange reactions, respectively. The analog binds tightly to the membrane-bound CF₁, competes with the right binding of ADP, and prevents the inactivation of the enzyme by tight binding of ADP. Upon irradiation with long wavelength ultraviolet light, the tightly bound BzADP becomes covalently attached to both the - and β-subunits of the enzyme.     

Engineered,highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side‐chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site‐specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100‐fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5‐cd]indolizine‐type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α‐helical, β‐sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q‐GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG‐reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.     

Photoaffinity labelling of the serotinin carrier protein in platelets and brain synaptosomes

Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focussing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [³H]azidoimipramine.     

Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling

Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.     

Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.     

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

Binding affinities and protein ligand complex geometries of nucleotides at the F(1) part of the mitochondrial ATP synthase obtained by ligand docking calculations

F₀F₁ ATP synthases utilize a transmembrane electrochemical potential difference to synthesize ATP from ADP and phosphate. In this work, the binding modes of ADP, ATP and ATP analogues to the catalytic sites of the F₁ part of the mitochondrial ATP synthase were investigated with ligand docking calculations. Binding geometries of ATP and ADP at the three catalytic sites agree with X-ray crystal data; their binding free energies suggest an assignment to the ‘tight’, ‘open’ and ‘loose’ states. The rates of multi-site hydrolysis for two fluorescent ATP derivatives were measured using a fluorescence assay. Reduced hydrolysis rates compared to ATP can be explained by the ligand docking calculations.