Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots.

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. FROM THE CLINICAL EDITOR: This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Advanced procedures for labeling of antibodies with quantum dots.

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.

Drug-eluting beads for liver embolization: concentration of doxorubicin in tissue and in beads in a pig model.

PURPOSE: To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests. MATERIALS AND METHODS: Left lobe hepatic artery embolization was performed in 10 pigs with 100-300-microm or 700-900-microm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100-300-microm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron-stained sections. RESULTS: Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time (P = .0004). The drug was detected at distances as far as 600 microm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 microM to 6.80 microM, [corrected] which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100-300 microm in size induced more necrosis than 700-900-microm beads (P = .0036). CONCLUSIONS: Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects.

High heterogeneity of plasma membrane microfluidity in multidrug-resistant cancer cells.

Diffusion-time distribution analysis (DDA) has been used to explore the plasma membrane fluidity of multidrug-resistant cancer cells (LR73 carcinoma cells) and also to characterize the influence of various membrane agents present in the extracellular medium. DDA is a recent single-molecule technique, based on fluorescence correlation spectroscopy (FCS), well suited to retrieve local organization of cell membrane. The method was conducted on a large number of living cells, which enabled us to get a detailed overview of plasma membrane microviscosity, and plasma membrane micro-organization, between the cells of the same line. Thus, we clearly reveal the higher heterogeneity of plasma membrane in multidrug-resistant cancer cells in comparison with the nonresistant ones (denoted sensitive cells). We also display distinct modifications related to a membrane fluidity modulator, benzyl alcohol, and two revertants of multidrug resistance, verapamil and cyclosporin-A. A relation between the distribution of the diffusion-time values and the modification of membrane lateral heterogeneities is proposed.

Serum albumin-alginate coated microspheres: role of the inner gel in binding and release of the KRFK peptide.

In continuation with our previous study using fluorescein-isothiocyanate (FITC)-Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4x10(5) L/mol or 8x10(5) L/mol for gelation with 5% or 20% CaCl(2), respectively). Binding site number doubled (from 2.6x10(-7) mol/mg to more than 5x10(-7) mol/mg) when microsphere size decreased from 450 microm to 100 microm. Binding sites were assumed to be located in the gel underneath the membrane.

Extracellular matrix proteins protect human HT1080 cells against the antimigratory effect of doxorubicin.

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.

Antioxidants and doxorubicin supplementation to modulate CD14 expression and oxidative stress induced by vitamin D3 and seocalcitol in HL60 cells.

1alpha,25-dihydroxyvitamin D3 (VD3) and the EB1089 analog are well known for their roles in the modulation of proliferation and the differentiation of several malignant cells. In addition, VD3 or EB1089 displayed a high disposal of oxidant features and the ability to cause release of reactive oxygen species (ROS). We attempted to enhance HL60 cell differentiation and to limit ROS generation, by the association of deltanoids with doxorubicin and the antioxidants catalase (CAT), superoxide dismutase (SOD) and N-acetyl cystein (NAC). Differentiation of HL60 cells into monocytic lineage was studied by expression of mRNA, protein CD14 and functional differentiation by the nitroblue tetrazolium assay. The 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) dye allowed to evaluate in situ ROS generation. When associated with 0.1 nM EB1089, 15 nM doxorubicin induced an increase of differentiated cell percentage from 29% to 87% and did not affect VD3-treated cells. The association with doxorubicin also induced a significant increase of ROS release (p<0.05) versus VD3 and EB1089-treated cells. These results correspond to additivity of individual effects of doxorubicin and deltanoids. Antioxidant agents (10 nM NAC, 50 U/ml SOD or 2000 U/ml CAT) were associated with 10 nM VD3 or 1 nM EB1089 for 72 h. Compared to VD3 and EB1089 treatments, associations with antioxidants induced a slight increase of differentiated cells and a significant increase of CD14 mRNA. The highest differentiation effect occurred in the case of the EB1089-NAC association. Antioxidants induced a decrease (p<0.05) in ROS release generated by VD3 or EB1089 near the level of untreated cells. Thus, antioxidant agents demonstrated a protective effect against VD3 and EB1089 oxidative cytotoxicity and an enhancement of the monocyte differentiation. Combinations of antioxidants with deltanoids could dissociate the oxidative stress and differentiation.

Detection of drug-resistance genes using single bronchoscopy biopsy specimens.

Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

Energy transfer to analyse membrane-integrated mitoxantrone in BCRP-overexpressed cells.

The binding and the diffusion of mitoxantrone (MTX) through the plasma membrane was performed by Förster resonance energy transfer (FRET) from the membrane fluorescent donor (4Di-10ASP) to the co-localized acceptor MTX. The MTX addition to living 4Di-10ASP-tagged cells resulted in the rapid quenching of the probe emission (1s), revealing the MTX binding to the outer leaflet. Then, a slower quenching (about 90s) occurred which corresponded to the MTX flip-flop into the inner leaflet. Changes of MTX integration into the plasma membrane were described in BCRP-overexpressed cells (HCT-116R) treated with (i) the BCRP inhibitor fumitremorgin C (FTC), (ii) cyclosporin A (CSA) and (iii) benzyl alcohol (BA). Treatments with FTC or CSA showed 80% and 40% higher flip-flop of MTX from the outer to the inner leaflet of HCT-116R cells. The addition of BA clearly increased the MTX integration into both outer and inner leaflets. Confocal fluorescence microscopy displayed that FTC, CSA and BA enhanced MTX accumulation in HCT-116R. In conclusion, Fumitremorgin C and agents modulating MTX accumulation resulted in higher MTX integration in the resistant cell membrane and could disrupt the membrane cohesion. This energy transfer method appears well-adapted to describe the drug diffusion through the plasma membrane of living cells.

Association between host species choice and morphological characters of main sensory structures of Culicoides in the Palaeartic region.

Culicoides (Diptera: Ceratopogonidae) serve as vectors of several mammalian and avian diseases, including bluetongue, Schmallenberg, African horse sickness, avian malaria and Oropouche. Host preference investigations are necessary to assess the transmission routes of vector-borne diseases and to inform mitigation strategies. A recent study examining the main sensory structures (palps and antennae) of Culicoides species suggests that they be classified as ornithophilic or mammalophilic according to their feeding habits. We analyzed Culicoides host preferences according to the literature and carried out a multiple correspondence analysis linking these preferences with morphological data. Seven out of 12 variables were found to be reliable predictors of host preference in Culicoides species: Antenna Flagellomer-Sensilla Coeloconica-Number: (7-10) and (11-13); Antenna Flagellomer-Sensilla Coeloconica IV-X: presence; Palpus-size: wide and/or narrow opening and shallow pit; Palpus-Shape: strongly swollen; Antenna-Short sensilla trichodea-distal part segment IV to X-Number: 2 seta each. Our results demonstrate that the presence of sensilla coeloconica and the maxillary palpus can be used to separate ornithophilic and mammalophilic or ornithophilic/mammalophilic species.

Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

In situ analysis of doxorubicin uptake and cytotoxicity in a 3D culture model of human HT-1080 fibrosarcoma cells.

In solid tumors, chemotherapeutics must adequately diffuse through the extracellular compartment to achieve their cytotoxic effect. Using quantitative microspectrofluorometry, both Doxorubicin penetration through three-dimensional (3D) collagen I matrices and its subsequent intranuclear accumulation into HT-1080 cells cultured in this microenvironment were directly assessed. Evidence that collagen delayed the Doxorubicin penetration for 1 h is presented. During that period, drug concentrations were lower in the nuclei in 3D compared to 2D matrices. Anthracyclines were also found to exhibit similar cytotoxicity in 2D and 3D after long term incubation. In conclusion, in this 3D culture model, collagen type I matrices delayed the early distribution of low molecular weight therapeutics and failed to affect their long-term cytotoxic effects, as previously reported. This model may provide a rationale for avoiding the emergence of intrinsic chemoresistance in tissue.

P-glycoprotein inhibitors modulate accumulation and efflux of xenobiotics in extra and intracellular Toxoplasma gondii.

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.

Modifications of cellular autofluorescence emission spectra under oxidative stress induced by 1 alpha,25dihydroxyvitamin D(3) and its analog EB1089.

We attempted to characterize the cellular autofluorescence phenomenon of living HL-60 cells and to appraise its modifications under oxidative stress conditions induced by 1 alpha,25(OH)(2)D(3) (VD(3)) and its analog EB1089. Autofluorescence emission spectra of human promyelocytic HL-60 leukemic cells were monitored using laser scanning confocal microspectrofluorometry under UV excitation. Evaluation of reactive oxygen species (ROS) release was performed using the 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) staining and fluorescence emission measurement. VD(3) (1, 10, 100 nM) or EB1089 (0.1, 1 and 10 nM) induces a decrease in autofluorescence emission intensity that can be attributed to the oxidation of the coenzyme nicotinamide adenine dinucleotide (phosphate) NAD(P)H into NAD(P)(+). A dose-dependent increase (p<0.05) in ROS release is observed in VD(3)- and EB1089-treated cells. As compared with VD(3)- or EB1089-treated cells, doxorubicin-VD(3) or doxorubicin-EB1089 treatments strongly decrease the autofluorescence intensity and induce a higher release of ROS (p<0.05). The association of antioxidants (N-acetyl cysteine, superoxide dismutase, catalase) with VD(3) or EB1089 induce a more limited autofluorescence decrease and a weaker ROS generation, as compared with VD(3) and EB1089 treated cells. In conclusion, the free radicals release, generated by VD(3) and EB1089, was associated with the decrease in autofluorescence emission and can be modulated by doxorubicin and antioxidants.

Inhibitory effects of extracellular Mg2+ on intracellular Ca2+ dynamic changes and thapsigargin-induced apoptosis in human cancer MCF7 cells.

The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8+/-0.3 to 1.6+/-0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 microM thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.

Selective interactions of ethidiums with G-quadruplex DNA revealed by surface-enhanced Raman scattering.

Complexes formed between G-quadruplex (G4)-conformed oligonucleotides and four ethidium derivatives were studied by surface-enhanced Raman spectroscopy (SERS) to detail the topology of complexes that support a G4 stabilization. Ethidium bromide (EB), which presents a weak ability to stabilize oligonucleotides in G4 conformation, displayed no SERS intensity modification when bound to G4, as compared with the free EB. Three ethidium derivatives have been selected due to their higher ability to stabilize G4 than EB. Bound with G4-conformed oligonucleotides, SERS intensity of these three ethidiums decreased by factors of about 6, 3.5, and 15. The high SERS quenching was interpreted as a loss of accessibility of silver colloids for G4-bound ethidiums. This could represent a new selective parameter useful to identify G4-stabilizing molecules. To apraise the role of the oligonucleotide sequence on the interaction mode, complexes were formed with eight G4-conformed oligonucleotides in which the three loops were either 5'-TTA-3' or 5'-AAA-3'. Spectra of ethidiums were sensitive to both lateral loops, opposite to the 3' and 5' G4 ends. The sequence of these loops are believed to be selective in the interaction mode of ethidiums for G4.

Binding of ATP to heat shock protein 90: evidence for an ATP-binding site in the C-terminal domain.

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181-37186). In this paper, the interactions of ATP with native hsp90 and its recombinant N-terminal (positions 1-221) and C-terminal (positions 446-728) domains were studied by isothermal titration calorimetry, scanning differential calorimetry, and fluorescence spectroscopy. Results clearly demonstrate that hsp90 possesses a second ATP-binding site located on the C-terminal part of the protein. The association constant between this domain of hsp90 and ATP-Mg and a comparison with the binding constant on the full-length protein are reported for the first time. Secondary structure prediction revealed motifs compatible with a Rossmann fold in the C-terminal part of hsp90. It is proposed that this potential Rossmann fold may constitute the C-terminal ATP-binding site. This work also suggests allosteric interaction between N- and C-terminal domains of hsp90.