NaYbF4@NaYF4 Nanoparticles: Controlled Shell Growth and Shape-Dependent Cellular Uptake.

This study presents a controlled synthesis of NaYbF4@NaYF4 core-shell upconversion nanoparticles using the hot-injection technique. NaYF4 shells with tunable morphologies including long-rod, short-rod, and quasi-sphere are grown on identical NaYbF4 core nanoparticles by controlled injection of acetate or trifluoroacetate precursors. Mechanistic investigations reveal that anisotropic interfacial strain accounts for the preferential growth of shell layers along the c-axis. However, the strain effect can be offset by the fast injection of shell precursors, leading to nearly isotropic growth of NaYF4 shells over the NaYbF4 core nanoparticles. The core-shell nanoparticles are further modified with DNA molecules and incubated with adenocarcinomic human alveolar basal epithelial cells. Based on a combination of characterizations by flow cytometry and confocal microscopy, favorable cellular uptake and DNA delivery are observed for the quasi-sphere nanoparticles, owing to the high dispersibility and easy membrane wrapping. The method described here could be extended to synthesize other types of functional nanostructures for the study of morphology-dependent properties.

Study on cytotoxicity, cellular uptake and elimination of rare-earth-doped upconversion nanoparticles in human hepatocellular carcinoma cells.

The growing use of rare-earth doped upconversion nanoparticles (UCNPs) has caused increasing concern about their biosafety. Here, to understand the toxicity of UCNPs and their mechanism in HepG2 cells, we systematically study the cytotoxicity, uptake and elimination behaviors of three types of UCNPs combined multiple cytotoxicity evaluation means with inductively coupled plasma mass spectrometry (ICP-MS) detection. Sodium yttrium fluoride, doped with 18% (molar ratio) ytterbium and 2% erbium (NaYF4: Yb3+, Er3+) was selected as the model UCNPs with two sizes (35 and 55 nm), and the poly(acrylic acid) and polyethylenimine were selected as the representatives of negative and positive surface coating of UCNPs, respectively. UCNPs were found to induce cytotoxicity in time- and dose-dependent manners, which might be mediated by reactive oxygen species generation and oxidative stress. Apoptosis, inflammation, and metabolic process were enhanced after cells exposed to 200 mg/L UCNPs for 48 h. Increase in the protein levels of cleaved caspased-9, cleaved caspase-3 and Bax and decrease in the anti-apoptotic protein, Bcl-2 suggested that the mitochondria mediated pathway was involved in UCNP-induced apoptosis. With the aid of ICP-MS, it demonstrated that the cytotoxicity was associated with internalized amount of UCNPs, which largely relied on their surface properties rather than size in the tested range. By comparing UCNPs with Y3+ ions, it demonstrated that NPs properties played a nonnegligible role in the cytotoxicity of UCNPs. These findings provide new insights for fundamental understanding of cytotoxicity of UCNPs and may contribute to more rational use of these materials in the future.

ZIF-8-Assisted NaYF4:Yb,Tm@ZnO Converter with Exonuclease III-Powered DNA Walker for Near-Infrared Light Responsive Biosensor.

This work reports a ZIF-8 (ZIF: Zeolitic Imidazolate Framework)-assisted NaYF4:Yb,Tm@ZnO upconverter for the photoelectrochemical (PEC) biosensing of carcinoembryonic antigen (CEA) under near-infrared (NIR) irradiation on a homemade 3D-printed device with DNA walker-based amplification strategy. The composite photosensitive material NaYF4:Yb,Tm@ZnO, as converter to transfer NIR import to photocurrent output, was driven from annealed NaYF4:Yb,Tm@ZIF-8. Yb3+ and Tm3+-codoped NaYF4 (NaYF4:Yb,Tm) converted NIR excitation into UV emission, matching with the absorption of ZnO for in situ excitation to generate the photocurrent. Upon target CEA introduction, the swing arm of DNA walker including the sequence of CEA aptamer carried out the sandwiched bioassembly with CEA capture aptamer on the G-rich anchorage DNA tracks-functionalized magnetic beads. Thereafter, DNA walker was triggered, and the swing arm DNA was captured by the G-rich anchorage DNA according to partly complementary pairing and Exonuclease III (Exo III) consumed anchorage DNA by a burnt-bridge mechanism to go into the next cycle. The released guanine (G) bases from DNA walker enhanced the photocurrent response on a miniature homemade 3D-printed device consisting of the detection cell, dark box, and light platform. Under optimal conditions, NaYF4:Yb,Tm@ZnO-based NIR light-driven PEC biosensor presented high sensitivity and selectivity for CEA sensing with a detection limit of 0.032 ng mL-1. Importantly, our strategy provides a new horizon for the development of NIR-based PEC biosensors in the aspect of developing MOF-derived photoelectric materials, flexible design of a 3D-printed device, and effective signal amplification mode.

Noninvasive and Reversible Cell Adhesion and Detachment via Single-Wavelength Near-Infrared Laser Mediated Photoisomerization.

Dynamically regulating cell-molecule interactions is fundamental to a variety of biological and biomedical applications. Herein, for the first time, by utilizing spiropyran conjugated multishell upconversion nanoparticles (UCNPs) as a new generation of single-wavelength near-infrared (NIR)-controlled photoswitch, we report a simple yet versatile strategy for controlling cell adhesion/detachment reversibly and noninvasively. Specifically, the two-way isomerization of the photoswitch was merely dependent on the excitation power density of the 980 nm laser. At high power density, the ring-opening was prominent, whereas its reverse ring-closing process occurred upon irradiation by the same laser but with the lower power density. Such transformations made the interactions between spiropyran and cell surface protein fibronectin switchable, thus leading to reversible cell adhesion and detachment. Moreover, efficient adhesion-and-detachment of cells could be realized even after 10 cycles. Most importantly, the utilization of NIR not only showed little damage toward cells, but also improved penetration depth. Our work showed promising potential for in vivo dynamically manipulating cell-molecule interactions and biological process.

Polyethylene glycol compared with ytterbium oxide as a total faecal output marker to predict organic matter intake of dairy ewes fed indoors or at pasture.

Several external markers can be used for estimating total faecal output in view of assessing ruminant intake at pasture. Among them, ytterbium (Yb) has been used for many years in various conditions. Polyethylene glycol (PEG) is a promising external marker because it can be rapidly determined using near-infrared spectroscopy (NIRS). The study consisted of 24 adult lactating dairy ewes over three periods (P1, P2 and P3), fed with three different diets: P1, total mixed ration (TMR); P2, Italian ryegrass (IRG); and P3, pasture. After an adaptation period, the ewes were administered a daily dose of ytterbium oxide (0.35 g/day) and PEG (20 g/day) for 2 weeks. During the last week, the daily organic matter intake (OMIOBS) was measured. Faecal samples were collected at milking time (0800 and 1600 h) to determine marker content, using only samples collected in the morning (PEGm) or by averaging samples (Yb, PEGma). Faecal marker content made it possible to assess total faecal output, either using the two recovery rates for PEG (0.98 or 0.87) or not. The OMIOBS was assessed on the basis of total faeces estimated with Yb (OMIYb) or PEG (OMIPEG), and the digestibility was calculated on the basis of feed analysis. With total TMR (P1), the OMIPEG, corrected with recovery rate (OMIPEGm98) or not corrected (OMIPEGm) was 2.40 kg/day and 2.50 kg/day, respectively, and was not different (P>0.05) from OMIOBS (2.51 kg/day), whereas OMIYb was lower (2.14 kg/day) (P<0.001). With IRG (P2), OMIPEGm98 (1.67 kg/day), OMIPEGm87 (1.51 kg/day) and OMIYb (1.59 kg/day) were not different (P>0.05) from OMIOBS (1.57 kg/day). With pasture (P3), the OMIPEGm (1.54 kg/day) and OMIPEGm98 (1.48 kg/day) were not different (P>0.05) from the OMI assessed from the biomass measurement (1.52 kg/day). The OMIYb (1.36 kg/day) was lower (P<0.05) but not different from OMIPEGm98 and OMIPEGm87. Spearman's rank correlation between OMIOBS and other OMIs (predicted with Yb or PEG P1 and P2) showed that it is possible to rank animals using PEG when there is a sufficiently wide range of OMIOBS (1.65 to 2.8 kg/day in P1) but not within a narrower range (1.47 to 1.72 kg/day in P2). In conclusion, the present study confirms that PEG is a valuable external faecal marker, easy to prepare (solution), administer and determine (NIRS). It can be used to assess intake with numerous animals at pasture, but only for groups, and not to quantitatively estimate individual OMI.

Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells.

An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er(3+) and Yb(3+) doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future 'smart' theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor.

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl(3) (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)(3). Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)(3) with ds-DNA. It was revealed that Yb(QS)(3) presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)(3) with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10(-8) to 1.1 × 10(-7)M. The interaction mode between Yb(QS)(3) and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.

[Research of biocompatibility of nanoparticles with fluorescent domen Er/Yb in the neutrophilic granulocytes system].

Fluorescent tags are extensively used for the diagnostic, labeling and marking in vivo and in vitro systems. In this study, fluorescent nanoparticles with Er/Yb lightning center were tested on neutrophilic granulocytes. The main purpose was to idenfity possible toxic effect. The negative impact of fluorophores on the metabolism of neutrophilic and their enzyme systems, on the receptor-mediated cell responses and on the rigidity of cell membranes was shown. The viability of neutrophils (estimated with the use of propidium iodide) after 2 hours of incubation with the fluorescent nanoparticles in concentrations of 10(-4) and 10(-3) mM was 27.0 +/- 6.6 and 19.07 +/- 3.34 %, respectively.

Ytterbium and trace element distribution in brain and organic tissues of offspring rats after prenatal and postnatal exposure to ytterbium.

Lanthanides, because of their diversified physical and chemical effects, have been widely used in a number of fields. As a result, more and more lanthanides are entering the environment and eventually accumulating in the human body. Previous studies indicate that the impact of lanthanides on brain function cannot be neglected. Although neurological studies of trace elements are of paramount importance, up to now, little data are provided regarding the status of micronutritional elements in rats after prenatal and long-term exposure to lanthanide. The aim of this study is to determine the ytterbium (Yb) and trace elements distribution in brain and organic tissues of offspring rats after prenatal and long-term exposure to Yb. Wistar rats were exposed to Yb through oral administration at 0,0.1, 2, and 40 mg Yb/kg concentrations from gestation day 0 through 5 mo of age. Concentrations of Yb and other elements (Mg, Ca, Fe, Cu, Mn, and Zn) in the serum, liver, femur, and brain regions (cerebral cortex, hippocampus, cerebellum, and the rest) of offspring rats at the age of 0 d, 25 d, and 5 mo were analyzed by inductively coupled plasma-mass spectrometry. The accumulation of Yb in the brain, liver, and femur is observed; moreover, the levels of Fe, Cu, Mn, Zn, Ca, and Mg in the brain and organic tissues of offspring rats are also altered after Yb exposure. This disturbance of the homeostasis of trace elements might induce adverse effects on normal physiological functions of the brain and other organs.

Synthesis and near infrared luminescence of a tetrametallic Zn2Yb2 architecture from a trinuclear Zn3L2 Schiff base complex.

Near infrared luminescence is observed in tetrametallic [Zn2Yb2L2(mu-OH)2Cl4].2MeCN which is obtained from the Zn3 Schiff-base complex [Zn3L2(NO3)2].MeOH, (H2L =N,N'-bis(5-bromo-3-methoxysalicylidene)propylene-1,3-diamine).

Biosorption of La, Eu and Yb using Sargassum biomass.

Biosorption of the lanthanides: Lanthanum (La(3+)), Europium (Eu(3+)) and Ytterbium (Yb(3+)) from single-component and multi-component batch systems using Sargassum polycystum Ca-loaded biomass was studied. The ion exchange sorption mechanism was confirmed by the release of calcium ions from the biomass that matched the total number of metal and protons removed from the solution. The metal binding increased with pH due to the decrease of proton concentration in the system, as they also compete for the binding sites. The maximum metal uptake capacity for pH 3, 4 and 5 ranged approximately between (0.8-0.9) mmol g(-1) for La (0.8-0.9) mmol g(-1) for Eu, and (0.7-0.9) mmol g(-1) for Yb. Biosorption from multi-component mixtures was examined at pH 4 using equimolar initial concentrations of the metals. The metal affinity sequence established was Eu>La>Yb, and the maximum metal uptake obtained was 0.29, 0.41, 0.28 mmol g(-1) for La, Eu and Yb, respectively.

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C.

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.

Binding affinity and capacities for ytterbium(3+) and hafinum(4+) by chemical entities of plant tissue fragments.

The binding affinity of ytterbium (Yb3+) and hafinum (Hf4+) to ligands of chemical entities of fragments of bermudagrass tissues and their resistance to exchanging Yb with other ligands and to displacement by protons were investigated. Chemical entities of acid resistant NDF (ARNDF), 0.1 N acid detergent fiber (0.1 N ADF), and permanganate cellulose (CELL) were prepared from fragments of bermudagrass hay (Cynodon dactylon [L.] Pers.) obtained by grinding to pass a 2-mm sieve. 175Ytterbium and Yb, as YbCl3, were initially bound to each preparation by soaking for 12 h in pH 5.5 borate buffer to obtain Yb bound onto ligands having affinity constants for Yb equal to or greater than that for the weakly stable borate ligand, Yb > or = borate. The fraction of Yb > or = borate was measured and fragments then sequentially exposed to acetate, citrate, nitrotriacetate (NTA), and EDTA ions to allow exchange of Yb from Yb > or = borate with ligands having affinity constants for Yb equal to or greater than acetate (Yb > or = acetate), citrate (Yb > or = citrate), NTA (Yb > or = NTA), and EDTA (Yb > or = EDTA) ions. Binding of Yb > or = borate indicated the existence of two species of ligands: strong ligands binding essentially 100% of added Yb at levels of 1 to 1,300 ppm (0.1 N ADF) and at 1 to 7,000 ppm (ARNDF); and weaker ligands binding 4 and 8% of the Yb, respectively, at levels of added Yb greater than 1,300 ppm and 7,000 ppm. Ytterbium > or = acetate of ARNDF, but not 0.1 N ADF, was as resistant to exchange as Yb > or = citrate. Ytterbium > or = borate was exchanged extensively (85% or greater) with soluble ligands having affinity constants > or = NTA. Ytterbium resistance to proton displacement at pH of 1.5 increased with Yb > or = EDTA > Yb > or = NTA > Yb > or = citrate > Yb > or = acetate. Very efficient binding of Yb to CELL suggested that such chemical preparations are not representative of native cellulose. Hafnium (4+) was strongly bound to plant tissues rendering both Hf and Hf-bound DM insoluble at a pH of 1.5 and insoluble in a modified NDF solvent without EDTA. It is concluded that Yb specifically applied as Yb > or = acetate and Hf4+ are indelible markers for estimating sojourn time of undigested plant tissues at the normal pH of the rumen. Because of its resistance to proton displacement, Hf4+ would be an indelible marker for estimating sojourn time in more acidic postgastric segments of the gastrointestinal tract.

The effect of feed intake on ileal rate of passage and apparent amino acid digestibility determined with or without correction factors in pigs.

The apparent ileal digestibilities of amino acids and rate of passage were evaluated in pigs (BW = 78.3 +/- 7.4 kg) fed a semipurified diet. The pigs were fed 1.82, 2.73, or 3.65 kg DMI/d. The highest level of feed intake was considered to be ad libitum feeding. The pigs were fed according to a 3 x 3 Latin square design and were allowed to adapt to each experimental diet for 5 d. This was followed by 1 d of continuous collection of ileal digesta and a 2nd d of continuous collection separated into six 2-h postprandial time blocks. Ytterbium chloride hexahydrate was used to determine rate of passage. The ileal digestibilities of amino acids and rate of passage were unaffected (P > 0.05) by level of feed intake. The use of correction factors to more accurately express amino acid concentrations in the diet and digesta affected (P < 0.05) the apparent ileal digestibility coefficients of some amino acids.

Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation.

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).

Non-covalent conjugates between cationic polyamino acids and GdIII chelates: a route for seeking accumulation of MRI-contrast agents at tumor targeting sites.

Three novel Gd chelates containing on their external surface pendant phosphonate and carboxylate groups, which promote the interaction with the positively charged groups of polyornithine and polyarginine, have been synthesized. Their solution structures have been assessed on the basis of 1H- and 31P-NMR spectra of the Eu and Yb analogues. A thorough investigation of the relaxometric (1H and 17O) properties of the Gd chelates has been carried out and the observed relaxivities have been accounted for the sum of three contributions arising from water molecules in the first, second, and outer coordination layers, respectively. It has been found that the occurrence of a tight second coordination coating renders the dissociation of the water molecule directly coordinated to the Gd ion more difficult. The binding interactions between the negatively charged Gd chelates and the positively charged groups of polyornithine (ca. 140 residues) and polyarginine (ca. 204 residues) have been evaluated by means of the proton relaxation enhancement (PRE) method. Although the binding interaction decreases markedly in the presence of competitive anions in the solution medium, the affinity is strong enough that in blood serum it is possible to meet the conditions where most of the chelate is bound to the polyamino acid substrate. On this basis one may envisage a novel route for a MRI location of tumors as it is known that positively charged polyamino acids selectively bind to tumors having a greater negative charge than non-tumor cells.

Adsorption of polyvalent cations to bilayer membranes from negatively charged lipid: estimating the lipid accessibility in the case of complete binding.

The effect of trivalent (Gd(3+) and Yb(3+)) and divalent (Be(2+) and Ca(2+)) cations on suspensions of multilamellar liposomes formed from brain PS and DMPS has been studied using microelectrophoresis and DSC techniques, respectively. The zeta potential values have been shown to strongly depend on the total lipid concentration in the suspension. At moderate concentrations of the polyvalent cations, the total cation concentration exceeds the bulk one several times due to adsorption of cations to the liposomes. A modification of the Gouy-Chapman-Stern theory in the case of unknown bulk concentration of the polyvalent cation is presented. An intrinsic association constant for Be(2+) ions was evaluated to be about K(2) approximately 50 M(-1). The algorithm for estimating the concentrations of the accessible (to exogenously added polyvalent cations) lipid-binding sites is described. These values are consistent with the subsurface concentrations of the polyvalent cations, which monotonously increase with the total concentration of the polyvalent cations. The calculated lipid accessibilities are shown to be in accordance with the DSC data.

Calcium binding to the photosystem II subunit CP29.

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.

Lanthanide induced residual dipolar couplings for the conformational investigation of peripheral 15NH2 moieties.

The Ca2 calbindin protein in which one calcium has been substituted with Ce(III), Yb(III) and Dy(III) displays substantial alignment in high magnetic fields due to the high anisotropy of the metal magnetic susceptibility. This property has allowed the measurement of residual dipolar coupling contributions to 1J(HN) and 2J(HH) couplings of asparagine and glutamine NH2 moieties. Such data have been used to aid structural characterization of these groups. The exploitation of auto-orientation of magnetic anisotropic metalloproteins represents a step ahead in the investigation of the conformational space of peripheral residues that are not fixed by the protein folding.