Conformational changes in the core structure of bacteriorhodopsin

Bacteriorhodopsin (bR) is the light-driven proton pump found in the purple membrane of Halobacterium salinarium. In this work, structural changes occurring during the bR photocycle in the core structure of bR, which is normally inaccessible to hydrogen/deuterium (H/D) exchange, have been probed. FTIR difference bands due to vibrations of peptide groups in the core region of bR have been assigned by reconstituting and regenerating delipidated bR in the presence of D2O. Exposure of bR to D2O even after long periods causes only a partial shift of the amide II band due to peptide NH --> ND exchange only of peripheral peptide structure. However, the amide II band completely downshifts when reconstitution/regeneration of bR is performed in the presence of D2O, indicating that almost the entire core backbone structure of bR undergoes H/D exchange. Peripheral regions can then be reexchanged in H2O, leaving the core backbone region deuterated. Low-temperature FTIR difference spectra on these core-deuterated samples reveal that peptide groups in the core region respond to retinal isomerization as early as the K intermediate. By formation of the M intermediate, infrared differences in the amide I region are dominated by much larger structural changes occurring in the core structure. In the amide II region, difference bands appear upon K formation and increase upon M formation which are similar to those observed upon the cooling of bacteriorhodopsin. This work shows that retinal isomerization induces conformational changes in the bacteriorhodopsin core structure during the early photocycle which may involve an increase in the strength of intramolecular alpha-helical hydrogen bonds.     

Transient channel-opening in bacteriorhodopsin: an EPR study

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.     

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

Photoproducts of bacteriorhodopsin mutants: a molecular dynamics study

Molecular dynamics simulations of wild-type bacteriorhodopsin (bR) and of its D85N, D85T, D212N, and Y57F mutants have been carried out to investigate possible differences in the photoproducts of these proteins. For each mutant, a series of 50 molecular dynamics simulations of the photoisomerization and subsequent relaxation process were completed. The photoproducts can be classified into four distinct classes: 1) 13-cis retinal, with the retinal N-H+ bond oriented toward Asp-96; 2) 13-cis retinal, with the N-H+ oriented toward Asp-85 and hydrogen-bonded to a water molecule; 3) 13,14-di-cis retinal; 4) all-trans retinal. Simulations of wild-type bR and of its Y57F mutant resulted mainly in class 1 and class 2 products; simulations of D85N, D85T, and D212N mutants resulted almost entirely in class 1 products. The results support the suggestion that only class 2 products initiate a functional pump cycle. The formation of class 1 products for the D85N, D85T, and D212N mutants can explain the reversal of proton pumping under illumination by blue and yellow light.     

Surface charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites

We present a method that allows the detection of the surface charge density of bacteriorhodopsin (bR) at any selected protein surface site. The optical pH indicator fluorescein was covalently bound to the sulfhydryl groups of single cysteine residues, which were introduced at selected positions in bR by site-directed mutagenesis. On the extracellular side, the positions were in the BC loop (72) and in the DE loop (129-134). On the cytoplasmic side, one position in each loop was labeled: 35 (AB), 101 (CD), 160 (EF), and 231 (carboxy tail). The apparent pKs of fluorescein in these positions were determined for various salt concentrations. The local surface charge density was calculated from the dependence of the apparent pK of the dye on the ionic strength using the Gouy-Chapman equation. The surface charge density at pH 6.6 is more negative on the cytoplasmic side (averaged over all positions, -2.5 +/- 0.2 elementary charges per bR) than on the extracellular side (average, -1.8 +/- 0.2 elementary charges per bR) with little variation along the surface. Since the experiments were performed with electrically neutral CHAPS/DMPC micelles, these values represent the charge present on bR itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy transfer in Tb~（3＋）,Yb~（3＋） codoped Lu_2O_3 near-infrared downconversion nanophosphors

Tb3+ and Yb3+ codoped Lu2O3 nanophosphors were synthesized by the reverse-strike co-precipitation method. The obtained Lu2O3:Tb3+,Yb3+ nanophosphors were characterized by X-ray diffraction (XRD) and photoluminescence (PL) spectra. The XRD results showed that all the prepared nanophosphors could be readily indexed to pure cubic phase of Lu2O3 and indicated good crystallinity. The Tb3+→Yb3+ energy transfer mechanisms in the UV-blue region in Lu2O3 nanophosphors were investigated. The experimental results showed that the strong visible emission around 543 nm from Tb3+ (5D4→7F5) and near-infrared (NIR) emission around 973 nm from Yb3+ (2F5/2→2F7/2) of Lu2O3:Tb3+,Yb3+ nanophosphors were observed under ultraviolet light excitation, respectively. Tb3+ could be effectively excited up to its 4f75d1 state and relaxed down to the 5D4 level, from which the energy was transferred cooperatively to two neighboring Yb3+. The Yb3+ concentration dependent luminescent properties and lifetimes of both the visible and NIR emissions were also studied. The lifetime of the visible emission decreased with the increase of Yb3+ concentration, verifying the efficient energy transfer from the Tb3+ to the Yb3+. Cooperative energy transfer (CET) from Tb3+ to Yb3+ was discussed as a possible mechanism for the near-infrared emission. When doped concentrations were 1 mol.% Tb3+ and 2 mol.% Yb3+, the intensity of NIR emission was the strongest.     

Characterization of the single tyrosine containing troponin C from lungfish white muscle. Comparison with several fast skeletal muscle troponin C's from fish species

Troponin C molecules from fast skeletal muscle of the following fish species (trout, whiting, lungfish, tilapia, and cod) have been purified to homogeneity. Upon binding of Ca2+ or Mg2+, lungfish troponin C is the only troponin C from fish white muscle to show the typical increase of tyrosine fluorescence emission quantum yield reported for rabbit fast skeletal muscle troponin C. The increase of tyrosine fluorescence signal occurring upon Ca2+ and Mg2+ titration of lungfish troponin C has been used to determine the corresponding affinity constants. With K(Ca) = 7.0 10(7) M-1 and K(Mg) = 3.6 10(3) M-1, the sites probed by the tyrosine residue of lungfish troponin C are typical of the COOH-terminal domain of fast skeletal troponin C's. The amino acid sequencing of the tyrosine containing tryptic peptides has allowed us to position the single tyrosine residue at position 7 in the Ca2+ binding loop of the third site, in identical position to Tyr109 of troponin C from rabbit fast skeletal muscle. Metal ion binding studies followed by intrinsic fluorescence or Tb3+ luminescence indicate that the conformation of the structural domain of lungfish troponin C with one metal ion bound is close to the physiological conformation of this domain.     

Arginine activity in the proton-motive photocycle of bacteriorhodopsin: solid-state NMR studies of the wild-type and D85N proteins

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the epsilon position, an upfield shift of 4 ppm is observed while the eta nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg-82 on the basis of the SSNMR investigations of the alkaline and neutral dark-adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of eta signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg-134. The results are discussed in relation to existing models of bR structure and function.     

Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha methyl-D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH = 6.9 and at 25 degrees C. The metal substituted Con A derivatives consisted of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 A away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93 degrees C which also appears following the addition of Cd2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol-1 at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0.41 kJ mol-1 K-1.     

Kinetic isotope effects reveal an ice-like and a liquid-phase-type intramolecular proton transfer in bacteriorhodopsin

The mechanism of the intramolecular proton transfer in the membrane protein bacteriorhodopsin (bR) is studied. The kinetic isotope effects after H/D exchange were determined for the individual photocycle reactions and used as an indicator. Significant differences in the kinetic isotope effects are observed between the intramolecular proton transfer on the release and the uptake pathways. The results suggest a fast intramolecular proton transfer mechanism in the proton release pathway, which is similar to the one proposed for ice, where the rate limiting step is the proton movement within the H bond. However, the reactions in the intramolecular proton uptake pathway occur in a mechanism similar to the one suggested for liquid water, where the rate limiting step is given by a rotational rearrangement of H bonded network groups. We propose that the experimental evidence for a proton wire mechanism given here for bacteriorhodopsin is of general relevance also for other proton transporting proteins.     

Metal ion activation of galactosyltransferase

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.     

Thermodynamics of binding of calcium, magnesium, and zinc to the N-methyl-D-aspartate receptor ion channel peptidic inhibitors, conantokin-G and conantokin-T

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 degreesC and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 microM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 microM and another of Kd = 311 microM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 microM and 1.1 microM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 microM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 microM, 10.2 microM, and 0.5 microM, respectively. Again, in the case of con-T, a weak (Kd = 410 microM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.     

A permanent ion binding site located between two gates of the Shaker K+ channel

K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter.     

Up-conversion luminescence of Tm3+/Yb3+ co-doped oxy-fluoride glasses

The oxy-fluorosilicate glasses co-doped with Yb3+/Tm3+ were prepared.The absorption spectra were recorded.The Tm3+ ion showed two absorption bands,with one at 774 nm due to 1G4→3H6 transition and the other at 667 nm due to 1G4→3F4 transition.The energy transfer between Tm3+ ion and Yb3+ ion and the up-conversion fluorescence of Tm3+ ion were investigated using 980 nm LD excitation.The results showed that the blue and red emissions were three-photon absorption processes corresponding with 1G4→3H6 and 1G4→3F4...     

Green, orange, and red upconversion luminescence in Nd3+/Yb3+: Cs2NaGdCl6 powders under 785 nm laser excitation

Nd3+:Cs2NaGdCl6 and Nd3+, Yb3+:Cs2NaGdCl6 polycrystalline powder samples were prepared by Morss method E. Under 785 nm semiconductor laser pumping, the upconversion luminescence of Nd3+ ions in Cs2NaGdCl6 was investigated at room temperature, and three upconversion emissions near 538 nm (Green), 603 nm (Grange), and 675 nm (Red) were observed and assigned to 4G7/2→4I9/2, (4G7/2→4I11/2; 4G5/2→4I9/2), and (4G7/2→4I13/2; 4G5/2→4I11/2), respectively. The dependences of these upconverted emissions on laser power and Nd3+ ion con-eentration were investigated, to explore the upconversion mechanism. The effect of doping Yb3+ ions on the upconversion luminescence of Nd3+ in Cs2NaGdCl6 was also studied under 785 nm laser excitation. The energy transfer processes were discussed as the possible mecha-nism for the above upconversion emissions.     

Quantitative kinetic model for photoassembly of the photosynthetic water oxidase from its inorganic constituents: requirements for manganese and calcium in the kinetically resolved steps,

The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.     

Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-type mannose-binding protein

A proline residue flanked by two polar residues is a highly conserved sequence motif in the Ca2+- and carbohydrate-binding site of C-type animal lectins. Crystal structures of several C-type lectins have shown that the two flanking residues are only observed to act as Ca2+ ligands when the peptide bond preceding the proline residue is in the cis conformation. In contrast, structures of the apo- and one-ion forms of mannose-binding proteins (MBPs) reveal that, when the Ca2+-binding site is empty, the peptide bond preceding the proline can adopt either the cis or trans conformation, and distinct structures in adjacent regions are associated with the two proline isomers. In this work, measurements of Ca2+-induced changes in intrinsic tryptophan fluorescence, and fluorescence energy transfer from tryptophan to Tb3+, reveal a slow conformational change in rat liver MBP (MBP-C) accompanying the binding of either Ca2+ or Tb3+. The Ca2+-induced increase in intrinsic tryptophan fluorescence shows biphasic kinetics: a burst phase with a rate constant greater than 1 s(-1) is followed by a slow phase with a single-exponential rate constant ranging from 0.01 to 0.05 s(-1) (36 degrees C) that depends on the concentration of Ca2+. Likewise, addition of EGTA to Ca2+-bound or Tb3+-bound MBP-C causes a decrease in intrinsic tryptophan fluorescence with biphasic kinetics consisting of a burst phase with a rate constant greater than 1 s(-1), followed by a slow phase with a single-exponential rate constant of 0.065 s(-1). In contrast, Tb3+ fluorescence produced by resonant energy transfer from MBP-C decreases in a single kinetic phase with a rate constant greater than 1 s(-1), implying that the slow change in tryptophan fluorescence monitors a conformational change that is not limited in rate by ion dissociation. The rate constants of the slow phases accompanying Ca2+ binding and release are strongly affected by temperature and are weakly accelerated by the prolyl isomerase cyclophilin. These data strongly suggest that the binding of either Ca2+ or Tb3+ to MBP-C is coupled to a conformational change that involves the cis-trans isomerization of a peptide bond. Fitting of the data to kinetic models indicates that, in the absence of Ca2+, the proline in approximately 80% of the molecules is in the trans conformation. The slow kinetics associated with cis-trans proline isomerization may be exploited by endocytic receptors to facilitate sorting of carbohydrate-bearing ligands from the receptor in the endosome.     

Elucidation of individual cytochrome P450 enzymes involved in the metabolism of clozapine

The three-dimensional structures of E. coli inorganic pyrophosphatase (PPase) and its complexes with Mn2+ in a high affinity site and with Mg2+ in high and low affinity sites determined by authors in 1994-1996 at 1.9-2.2 A resolution are compared. Metal ion binding initiates the shifts of alpha-carbon atoms and of functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule. As a result, the apoPPase with six equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by structure and occupance of the low affinity Mg2+ site. Induced asymmetry reflects the subunit interactions and cooperativity between Mg2+ binding sites. These molecular rearrangements are structural basis to account for special features of the enzyme behavior and to propose one of the pathways for enzymatic activity regulation of constitutive PPases in vivo.