A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction.

Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl- dependent serotonin uptake, with estimated Km and Vmax values of 436 +/- 19 nm and 3.8 +/- 0.6 x 10-18 mol.cell.min-1, respectively. The Manduca high affinity Na+/Cl- dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 micro m) and cocaine (IC50 = 12.89 micro m). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148-152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 micro m). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1-146/MasSERT106-587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 micro m) compared to the human (IC50 = 0.431 micro m) and Manduca serotonin transporters. The chimera MasSERT1-67/hSERT109-630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 micro m) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.     

E. coli MEP synthase: steady-state kinetic analysis and substrate binding.

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.     

Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3beta-(4-methylphenyl)tropane-2beta-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); K(I) = 14 nm) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2beta-carbomethoxy-3beta-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (lambda(max)) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambda(max) of RTI-233 in aqueous solution and corresponding to the lambda(max) of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (K(SV =) 1.7 m(-)(1)), although not to the same extent as free RTI-233 (K(SV =) 7.2 m(-)(1)). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.     

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., ³H or ¹²⁵I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT K_i?=?0.16?nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT K_i?=?1.77?nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).     

Protein degradation by peroxide catalyzed by chromium (III): role of coordinated ligand

In order to understand the role of coordinated ligands in controlling the biotoxicity of chromium (III), interactions of three types of chromium (III) complexes viz. trans-diaquo [1,2 bis (salicyledeneamino) ethane chromium (III) perchlorate, [(Cr(salen)(H(2)O)(2)](ClO(4)); tris (ethylenediamine) chromium (III) chloride, [Cr(en)(3)]Cl(3), and monosodium ethylene diamine tetraacetato monoaquo chromiate (III), [Cr(EDTA)(H(2)O)]Na with BSA has been investigated. Spectroscopic and equilibrium dialysis studies show that the two cationic complexes Cr(salen)(H(2)O)(+)(2) and Cr(en)(3+)(3) bind to the protein with a protein-metal ratio of 1:8 and 1:4. The anionic complex Cr(EDTA)(H(2)O)(-) binds to the protein with a protein-metal ratio of 1:2. The binding constant K(b) as estimated from the fluorescence quenching studies has been found to be 7.6 +/- 0.4 x 10(3) M(-1), 3.1 +/- 0.2 x 10(2) M(-1), and 1.8 +/- 0.2 x 10(2) M(-1) for Cr(salen)(H(2)O)(+)(2), Cr(en)(3+)(3), and Cr(EDTA)(H(2)O)(-) respectively indicating that the thermodynamic stability of protein-chromium complex is Cr(salen)(H(2)O)(+)(2) > Cr(en)(3+)(3) approximately Cr(EDTA)(H(2)O)(-). The complexes Cr(salen)(H(2)O)(+)(2) and Cr(EDTA)(H(2)O)(-) in the presence of hydrogen peroxide have been found to bring about protein degradation, whereas Cr(en)(3+)(3) does not bring about any protein damage. This clearly shows that the nature of the chromium (III) complex plays a major role in the biotoxicity of chromium (III).     

Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. We determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [14C]iodoacetamide or enzymatic activity measurements. We estimated the distribution of the IANBD on the fiber proteins to be approximately 77% on SH2, approximately 5% on SH1, and approximately 18% on troponin I. We characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization [Ajtai, K., & Burghardt, T. P. (1987) Biochemistry 26, 4517-4523]. With wavelength-dependent fluorescence polarization we use the ability to rotate the transition dipole in the molecular frame using excitation wavelength variation to investigate the three angular degrees of freedom of the cross-bridge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ternary Cu(II) complexes, Cu(H(-1)L)(ACys-) and Cu(H(-2)L)(ACys-); L = peptides, ACys- = N-acetyl-cysteinate. Analogous complexes to the intermediates in the transport of Cu(II) from Cu(H(-2)L) to cysteine

The Cu(II) in Cu(H(-2)L) has been postulated to be successively transported to cysteine (Cys) as follows; Cu(H(-2)L) <==> Cu(H(-2)L)(Cys*-) <==> Cu(H(-1)L)(Cys*-) --> Cu(H(-1)L)(Cys-), where Cys*- denotes the monodentate Cys-. N-acetyl-cysteinate (ACys-) complexes Cu(H(-2)L)(ACys-) and Cu(H(-1)L)(ACys-), having similar coordination modes to Cu(H(-2)L)(Cys*-) and Cu(H(-1)L)(Cys*-), respectively, exhibited the S --> Cu(II) charge transfer absorption at 325-355 nm and the d-d absorption at 530-610 nm. A linear interrelation existed between the energies of the CD and d-d absorptions. Cu(H(-2)L)(ACys-) were in rapid equilibrium with Cu(H(-1)L)(ACys-). Upon forming the ternary complex, pK(c2) of the parent Cu(H(-1)L) was raised to more than 1.0. The formation constants (K) of the Cu(H(-1)L)(ACys-) species from Cu(H(-1)L) were bigger than those of Cu(H(-2)L)(ACys-) from Cu(H(-2)L). The linear free-energy relationship existed between the free-energy change (deltaG) and the entropy change (deltaS) for the ternary complex formation. The rate constants (k1+) for the Cu(H(-1)L)(Cys-) formation closely correlated with the K values for Cu(H(-2)L)(ACys-). The ternary complexes containing ACys are considered to be analogous complexes to the intermediates in the transport of Cu(II) from peptides to cysteine.     

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.     

Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.     

Enzymatic characterization and inhibitor discovery of a new cystathionine {gamma}-synthase from Helicobacter pylori

Cystathionine gamma-synthase (CGS) catalyses the first step of the transsulfuration pathway that converts l-cysteine to l-homocysteine in bacteria, whereas this pathway is absent in human. In this report, we identified a new metB gene from Helicobacter pylori strain SS1, and the recombinant H. pylori Cystathionine gamma-synthase (HpCGS) was successfully cloned, expressed and purified in Escherichia coli system. Enzymatic study of HpCGS indicated that the K(m) and k(cat)/K(m) values against the substrate O-succinyl-l-homoserine (l-OSHS) were 3.02 mM and 98.7 M(-)(1)s(-)(1), respectively. Moreover, four natural products (alpha-lapachone, 9-hydroxy-alpha-lapachone, Paulownin and Yangambin, Fig. 1) were discovered to demonstrate inhibitory activities against HpCGS with IC(50) values of 11 +/- 3, 9 +/- 1, 19 +/- 2 and 27 +/- 6 microM, respectively. All these four inhibitors prevent the binding of l-OSHS to HpCGS in a non-competitive fashion. In vitro antibacterial assays further indicated that these four discovered compounds could highly inhibit the growth of H. pylori and exhibited strong inhibitory specificity against H. pylori related to E. coli.     

Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors

Zinc binding to the two Cys(4) sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co(2+) as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys(4) structural zinc-binding sites found in the DBD of human estrogen receptor alpha (hERalpha-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co(2+) to the two proteins was determined relative to the binding affinity of Co(2+) to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co(2+) from CP-1, the dissociation constants of cobalt from hERalpha-DBD were calculated: K(d1)(Co) = 2.2 (+/- 1.0) x 10(-7) M and K(d2)(Co) = 6.1 (+/- 1.5) x 10(-7) M. Similarly, the dissociation constants of Co(2+) from GR-DBD were calculated: K(d1)(Co) = 4.1 (+/- 0.6) x 10(-7) M and K(d2)(Co) = 1.7 (+/- 0.3) x 10(-7) M. Metal-binding studies conducted in which Zn(2+) displaces Co(2+) from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn(2+) binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co(2+) does: the stoichiometric dissociation constants are K(d1)(Zn) = 1 (+/- 1) x 10(-10) M and K(d2)(Zn) = 5 (+/- 1) x 10(-10) M for hERalpha-DBD and K(d1)(Zn) = 2 (+/- 1) x 10(-10) M and K(d2)(Zn) = 3 (+/- 1) x 10(-10) M for GR-DBD. These affinities are comparable to those observed for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERalpha-DBD and GR-DBD do not interact.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

Characterization of Cdr1p, a major multidrug efflux protein of Candida albicans: purified protein is amenable to intrinsic fluorescence analysis

Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.     

Thermodynamics of the reduction of NADP with 2-propanol catalyzed by an NADP-dependent alcohol dehydrogenase

The apparent equilibrium constant of the biochemical reaction, 2-propanol+NADP(ox) = acetone+NADP(red), was determined at I = 0.25 M over a wide range of pH (5.63 to 8.02) and temperature (5 to 40 degrees C). The reaction was catalyzed by an NADP-dependent alcohol dehydrogenase. The results were used to calculate thermodynamic quantities for the chemical (ionic) reference reaction: 2-propanol+NADP(ox)(3-) = acetone+NADP(red)(4-)+H(+). The thermodynamic quantities for this reference reaction are as follows: equilibrium constant K = (5.98+/-0.46) x 10(-10); standard molar Gibbs energy change Delta(r)G(0) = (52.65+/-0.19) kJmol(-1); standard molar enthalpy change Delta(r)H(0) = (38.9+/-0.6) kJmol(-1); and standard molar entropy change Delta(r)S(0) = -(46.1+/-2.2)J K(-1)mol(-1). All of these results pertain to 25 degrees C (298.15 K) and I = 0. The results also lead, in conjunction with tabulated thermodynamic quantities, to the standard electromotive force E(0) = -0.140 V for the reduction of NADP(ox)(3-) to NADP(red)(4-).     

Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [(188)Re(H(2)O)(3)(CO)(3)](+) and synthesis of tailor-made bifunctional ligand systems

Two kit preparations of the organometallic precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in aqueous media are presented. Method A uses gaseous carbon monoxide and amine borane (BH(3).NH(3)) as the reducing agent. In method B CO(g) is replaced by K(2)[H(3)BCO(2)] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 degrees C. HPLC and TLC analyses revealed 7 +/- 3% of unreacted (188)ReO(4)(-) and <5% of colloidal (188)ReO(2). Solutions of up to 14 GBq/mL Re-188 have been successfully carbonylated with these two methods. The syntheses of two tailor-made bifunctional ligand systems for the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) are presented. The tridentate chelates consist of a bis[imidazol-2-yl]methylamine or an iminodiacetic acid moiety, respectively. Both types of ligand systems have been prepared with alkyl spacers of different length and a pendent primary amino or carboxylic acid functionality, enabling the amidic linkage to biomolecules. The tridentate coordination of the ligands to the rhenium-tricarbonyl core could be elucidated on the macroscopic level by X-ray structure analyses and 1D and 2D NMR experiments of two representative model complexes. On the nca level, the ligands allow labeling yields >95% with [(188)Re(H(2)O)(3)(CO)(3)](+) under mild reaction conditions (PBS buffer, 60 degrees C, 60 min) at ligand concentrations between 5 x 10(-4) M and 5 x 10(-5) M. Thus, specific activities of 22-220 GBq pe micromol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 degrees C revealed stabilities between 80 +/- 4% and 45 +/- 10% at 24 h, respectively, and 63 +/- 3% and 34 +/- 3% at 48 h postincubation in human serum depending on the chelating system. Decomposition product was mainly (188)ReO(4)(-). The routine kit-preparation of the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in combination with tailor-made ligand systems enables the organometallic labeling of biomolecules with unprecedented high specific activities.     

Remarkably stereoselective photoinduced electron-transfer reaction between zinc myoglobin and optically active binaphthyl bisviologen

We have designed and synthesized new optically active bisviologens ([BNMV](4+)) containing a binaphthyl moiety to examine the stereoselective photoinduced electron-transfer (ET) reactions with zinc-substituted myoglobin (ZnMb) by flash photolysis. The photoexcited triplet state of ZnMb, (3)(ZnMb)*, was successfully quenched by [BNMV](4+) ions to form the radical pair of a ZnMb cation (ZnMb(.+)) and a reduced viologen ([BNMV](.3+)), followed by a thermal ET reaction to the ground state. The rate constants ( k(q)) for the ET quenching at 25 degrees C were obtained as k(q)( R)=(2.9+/-0.2)x10(7) M(-1) s(-1) and k(q)( S)=(2.2+/-0.2)x10(7) M(-1) s(-1), respectively. The ratio of k(q)( R)/ k(q)( S)=1.3 indicates that the ( R)-isomer of the chiral viologen preferentially quenches (3)(ZnMb)*. On the other hand, the rate constants ( k) for the thermal ET reaction from [BNMV](.3+) to ZnMb(-+) at 25 degrees C were k( R)=(1.2+/-0.1)x10(8) M(-1) s(-1) and k( S)=(0.47+/-0.03)x10(8) M(-1) s(-1), respectively, and the ratio remarkably increased to k( R)/ k( S)=2.6. The activation parameters, Delta H(not equal) and Delta S(not equal), were determined from the kinetic measurements at various temperatures (10-30 degrees C) to understand the ET mechanisms. In the quenching reaction, the energy differences of Delta Delta H*(R- S) and T Delta Delta S*( R- S) at 25 degrees C were calculated to be -3.9+/-1.6 and -3.3+/-0.2 kJ mol(-1), respectively, whereas Delta Delta H*( R-S)=7.7+/-1.9 kJ mol(-1 )and T Delta Delta S*( R-S)=9.9+/-0.5 kJ mol(-1 )were found for the thermal ET reaction. Therefore, the thermal ET reaction to the ground state was proved to be dominated by the entropy term, and the large stereoselectivity may arise from the decrease in charge repulsion between donor and acceptor.     

Serotonin and cocaine-sensitive inactivation of human serotonin transporters by methanethiosulfonates targeted to transmembrane domain I

To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4 degrees C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.     

Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.     

Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/- 20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.