Design of a photoaffinity label for the hormone binding site of neurophysin.

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.     

Time-resolved infrared spectroscopy of electron transfer in bacterial photosynthetic reaction centers: dynamics of binding and interaction upon QA and QB reduction.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [M?ntele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [M?ntele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; M?ntele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., M?ntele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., M?ntele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modelling a binuclear metal binding site in the photosynthetic reaction centre II

Based on the experimental data and homologous sites in Protein Data Bank (PDB) a model for metal binding sites in D1/D2 heterodimer has been proposed. On searching for tetranuclear and binuclear Mn binding sites in the PDB, a suitable sequence homology in thermolysin and D1 could be observed. From the homology and site-directed mutagenesis data, a model for binuclear Mn-Ca or Mn-Mn has been built and it is extended to a tetranuclear Mn centre.     

2-Nitroimipramine: a photoaffinity probe for the serotonin uptake/tricyclic binding site complex.

[3H]2-Nitroimipramine ([3H]2-NI), a compound with high affinity for the serotonin uptake system, is shown to be an effective photoaffinity probe which incorporates covalently into membrane homogenates prepared from human platelets, as well as rat brain and liver. In all cases, [3H]2-NI preferentially incorporated into a minor membrane component of 30 kd protein, as determined by SDS-polyacrylamide gel electrophoresis and subsequent fluorography. A number of selective and general serotonin uptake inhibitors quantitatively chased labeling of the 30-kd band at nanomolar concentrations. Pharmacological characterizing agents unrelated to the serotonin uptake system generally had little effect on labeling. In platelet membranes, a broad band of approximately 35-kd protein was also labeled by [3H]2-NI, but this labeling was not inhibited by any of the selective serotonin uptake blockers. Interestingly, serotonin itself increased incorporation into the 30-kd band and selectively decreased labeling of the 35-kd band. Photolytic incorporation into the 30-kd band was of high affinity, saturable, and Scatchard analyses of irreversible labeling were linear. In contrast, Scatchard transformations of [3H]2-NI equilibrium binding saturation isotherms were markedly curvilinear. Cross-linking unlabeled 2-NI to intact platelets, followed by extensive dialysis, decreased the maximal velocity (Vmax) of platelet serotonin uptake, but did not alter the affinity (Km) of serotonin for its transport site. These results are noteworthy since current theories implicate prejunctional allosteric interactions between serotonin and imipramine at serotonergic synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of azidobestatin as a photoaffinity label to identify the active site peptide of leucine aminopeptidase.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. They are found in most cells and tissues, and their activity has been implicated in myriad fundamental biochemical and physiological processes. Nevertheless, little is known about the structure of the aminopeptidase active sites. Beef lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. Bestatin, [(2S,3R)-(3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] is a nonhydrolyzable substrate analogue of a peptide, PheLeu, which is rapidly cleaved by blLAP. Bestatin incorporates elements of the putative tetrahedral intermediate, and this results in a greater than 10(5)-fold enhancement of binding relative to analogous peptides. Bestatin is the most tightly bound inhibitor of many aminopeptidases. Bestatin was successively converted to nitrobestatin, p-aminobestatin, [3H]-p-aminobestatin, and finally [3H]-p-azidobestatin (pAB). Like bestatin, pAB is a slow binding inhibitor of LAP (Ki*, the dissociation constant for the final complex, = approximately 4 x 10(-9); Ki, the dissociation constant for the initial collision complex, = approximately 10(-8). The t1/2 for binding of 2 x 10(-8) M and 8 x 10(-8) M bestatin are approximately 60 min and approximately 38 min, respectively. pAB, nitrobestatin, bestatin, and physiological peptides appear to bind in the same site, the first three with similar avidity. In the dark, pAB and bestatin protect low concentrations of the enzyme against inactivation upon extensive dialysis. The t1/2 for photoactivation of pAB is approximately 3 s. Irradiation of blLAP for such short periods of time resulted in insignificant change in activity. blLAP which was placed in 254-nm light in the presence of pAB was inactivated significantly. Treatment of photolabeled blLAP with trypsin produces only two peptides. Autoradiography and scintillation counting indicate that the active site is in the peptide which includes residues 138-487. Treatment of the same blLAP with hydroxylamine produces two different peptides, with the active site in the peptide 323-487. This indicates that the active site is in the carboxyl-terminal one-third of the protomer. It is likely that this photoaffinity label will be useful in identifying active sites in other aminopeptidases as well.     

An x-ray absorption study of the iron site in bacterial photosynthetic reaction centers.

Measurements were made of the extended x-ray absorption fine structure (EXAFS) of the iron site in photosynthetic reaction centers from the bacterium Rhodopseudomonas sphaeroides. Forms with two quinones, two quinones with added o-phenanthroline, and one quinone were studied. Only the two forms containing two quinones maintained their integrity and were analyzed. The spectra show directly that the added o-phenanthroline does not chelate the iron atom. Further analysis indicates that the iron is octahedrally coordinated by nitrogen and/or oxygen atoms located at various distances, with the average value of about 2.14 A. The analysis suggests that most of the ligands are nitrogens and that three of the nitrogen ligands belong to histidine rings. This interpretation accounts for several unusual features of the EXAFS spectrum. We speculate that the quinones are bound to the histidine rings in some manner. Qualitative features of the absorption edge spectra also are discussed and are related to the Fe-ligand distance.     

Identification of a putative sordarin binding site in Candida albicans elongation factor 2 by photoaffinity labeling

Candida albicans EF-2 binds sordarin to a single class of binding sites with K(d) = 1.26 microm. Equimolar mixtures of EF-2 and ribosomes, in the presence of a non-hydrolyzable GTP analog, reveal two classes of high affinity sordarin binding sites with K(d) = 0.7 and 41.5 nm, probably due to the existence of two ribosome populations. Photoaffinity labeling of C. albicans EF-2 in the absence of ribosomes has been performed with [(14)C]GM258383, a photoactivatable sordarin derivative. Labeling is saturable and can be considered specific, because it can be prevented with another sordarin analog. The fragment Gln(224)-Lys(232) has been identified as the modified peptide within the EF-2 sequence, Lys(228) being the residue to which the photoprobe was linked. This fragment is included within the G"-subdomain of EF-2. These results are discussed in the light of the high sordarin specificity toward fungal systems.     

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.     

Expression of functionality of alpha-chymotrypsin. An alternate binding mode in the substrate specificity site

Three-dimensional 2.8 A resolution x-ray crystallographic studies show that toluenesulfonamide and pipsylamide bind isomorphously in the aromatic specificity binding site of alpha-chymotrypsin. However, their orientation differs by about 90 degrees from that usually associated with substrate-like molecules, suggesting a nonproductive binding mode. A secondary binding site is also operative in one molecule of the dimer of the pipsylamide derivative and it is located some 22 A from the active site; however, this site is not operative in the toluenesulfonamide derivative. Binding of toluenesulfonamide and pipsylamide in the specificity site occurs without inducing any significant changes in the native enzyme structure, in contrast to the behavior observed upon tosylation or upon transition state analogue binding of phenylethaneboronic acid. The structural changes accompanying the formation of the latter derivatives are generally asymmetric with respect to the dimeric structure of alpha-chymotrypsin and are generally confined to the binding domain or cylinder 2 of the enzyme (sequence greater than 122). These changes are displayed in a new way via diagonal distance map representation.     

Effect of visible light on the mitochondrial inner membrane.

Exposure of mitochondria to visible light in the presence of riboflavin resulted in the initial release of respiratory control, followed by inhibition of electron transport and dissolution of structural integrity. Under these conditions, however, cytochrome c oxidase activity remained unchanged. ATPase activity was stimulated initially and remained in this activitated state even under continued illumination. In submitochondrial preparations, both electron transport and ATPase declined as a function of illumination time; cytochrome c oxidase was not sensitive to light. Enzyme inactivation also occurred to a lesser extent in the absence of riboflavin.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers

Photosynthesis Research - The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone....     

Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol.

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.     

On the depletion and reconstitution of both QA and metal in reaction centers of the photosynthetic bacterium Rb. sphaeroides R-26

Four possible ways to prepare Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs were studied: (1) first depleting the Fe, then depleting Q_A and finally reconstituting Q_A (D-Fe, D-Q, R-Q), (2) first depleting Q_A, then depleting the Fe and finally reconstituting Q_A (D-Q, D-Fe, R-Q), (3) first depleting Q_A, then reconstituting Q_A and finally depleting Fe (D-Q, R-Q, D-Fe), (4) first depleting Q_A, then depleting the Fe and reconstituting Q_A in the same step (D-Q, D-Fe-R-Q). Our results showed that: method (1) results in the irreversible loss of photochemical activity; method (2) and (3) result in low recovery of the photochemical activity and poor yield of Fe-depleted, Q_A-reconstituted RCs; method (4) gives surprisingly good results. This method allows for the first time to prepare the Q_A-depleted, Fe-depleted, Q_A-reconstituted RCs with high recovery of the photochemical activity and good yield. The sample has 98% of photochemical activity (yield of P⁺ Q_A ^-) compared with that of the native RCs and shows strong polarization of the EPR signal of Q_A ^- under continuous illumination at 5K. The decay halftime of I^- is slow (5 ns) compared with that of the native RCs, but it is the same as that measured for the RCs from which only iron is removed. These results indicate that the depletion of iron and the reconstitution of Q_A have been successful. Reconstitution of the Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs with Zn²⁺ gives also the spin-polarized Q_A ^-, and yields the same decay of I^- (halftime 200 ps) as that of the native RCs.Abbreviations LDAO lauryldimethylamine N-oxide - EDTA ethylenediaminetetraacetic acid - BSA albumin bovine - TL buffer 10 mM Tris.HCl, 0.1% LDAO and 0.1 mM EDTA