Synthetic procedures to monomethyl-platinum(II) complexes containing nitrogen ligands of biological relevance

The complex trans-bis(dimethylsulfoxide)chloromethylplatinum(II) (1) is fairly soluble in water, where it undergoes multiple equilibria involving the formation of geometrically distinct [Pt(H(2)O)(DMSO)Cl(CH(3))] aqua-species. On reacting an aqueous solution of 1 with monodentate nitrogen donor ligands L, such as pyridines or amines, two well distinct patterns of behavior can be recognized: (i) a single stage fast substitution of one DMSO by the entering ligand, yielding a complex of the type trans(C,N)-[Pt(DMSO)(L)Cl(CH(3))] which contains four different groups coordinated to the metal and which undergoes a slow conversion into its cis-isomer, (ii) a double substitution affording cationic complex ions of the type cis-[Pt(L)(2)(DMSO)(CH(3))](+). When this latter reaction is carried out using sterically hindered ligands, slow rotation of the bulk ligand around the Pt[bond]N bond allows for the identification of head-to-head and head-to-tail rotamers in solution, through (1)H NMR spectrometry. The addition of chloride anion to 1 leads to the anionic species cis-[Pt(DMSO)Cl(2)(CH(3))](-), where a molecule of DMSO still remains coordinated to the metal center, despite its quite fast rate of ligand exchange (k(exch) with free DMSO=12+/-1 s(-1)). The reaction of complex 1 with bidentate ligands, such as ethylenediamine (en) or simple amino acids, leads to the cationic species [Pt(en)(DMSO)(CH(3))](+) or to the neutral [Pt(DMSO)(N[bond]O)(CH(3))], (where N[bond]-O[double bond]GlyO(-), AlaO(-)).     

Self-assembly of dendrimer-encapsulated nanoparticle arrays using 2-D microbial S-layer protein biotemplates

We investigated the formation of self-assembled two-dimensional (2-D) arrays of dendrimer-encapsulated platinum nanoparticles (Pt-DENs) using prokaryotic surface-layer (S-layer) proteins as biomacromolecular templates. The Pt-DENs (mean core diameter 1.8 +/- 0.5 nm) were synthesized by chemical reduction of metal ion species complexed within the interior of fourth-generation, hydroxyl-terminated, starburst poly(amidoamine) dendrimers (G4 PAMAM-OH). Detailed structural and elemental composition analyses performed using high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and X-ray photoelectron spectroscopy indicated that the dendrimer-metal nanocomposite particles were crystalline in nature rather than amorphous and that at least some quantity of the platinum found within the particles is present in the expected zerovalent state. By using the S-layer lattices from the acidothermophilic archaeon Sulfolobus acidocaldarius and the Gram-positive bacterium Deinococcus radiodurans as a biotemplate, hexagonal- and honeycomb-ordered arrays of the Pt-DENs were successfully fabricated under a range of different pH conditions via noncovalent nanoparticle-protein interactions. Fast Fourier transform analyses of transmission electron microscopy images verified that the fabricated Pt-DEN assemblies displayed mean periodicities that corresponded well with the lattice constants of the native protein templates (i.e., 22 and 18 nm for S. acidocaldarius and D. radiodurans S layers, respectively). Our results demonstrate that utilizing pre-synthesized Pt-DENs in conjunction with microbial S-layer proteins displaying highly periodic topochemical properties can be an effective, novel route for creating patterned arrays of Pt nanoparticles with potential technological applications.     

Reaction of DNA oligonucleotides with [Pt(dien)GSMe]2+ (GSMe =S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+: evidence of oligonucleotide platination via sulfur-coordinated platinum intermediates

To study the possibility of DNA platination via platinum-sulfur coordinated intermediates, the reactions of the complexes [Pt(dien)GSMe]2+ (GSMe=S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+ with the synthetic oligonucleotides d(ATATGCATAT), d(ATTACCGGTAAT), and d(ATCCTATTTTTTTTAGGAT) have been investigated. The reactions were studied using FPLC, NMR, and mass spectrometry. It was found that the sulfur atom of the platinum-thioether adduct is substituted by these oligonucleotides. For the reactions with [Pt(dien)GSMe]2+ at 310 K, half-lives were determined to be t 1/2 =147+/-7 h for d(ATATGCATAT), t 1/2 =84+/-4 h) for d(ATTACCGGTAAT), and t 1/2 = 21+/-1 h for d(ATCCTATTTTTTTTAGGAT. This study clearly shows that it is indeed possible for oligonucleotides to be platinated via Pt-thioether coordinated intermediates. The rates at which such substitutions occur, however, makes it improbable that such a mechanism contributes significantly to the antitumor activity of cisplatin.     

Antiproliferative activity of Pt(II) and Pd(II) phosphine complexes with thymine and thymidine

Oxidative addition reactions between [M(PPh(3))(4)] (M=Pt and Pd) and N1-methylthymine (t)/3',5'-di-O-acetylthymidine (T) were carried out to give [M(II)(PPh(3))(2)Cl t (or T)] complexes, in which the metal is coordinated to the N3 of the base. All complexes were characterized by spectroscopic analyses (IR, NMR) and Fast Atom Bombardment mass spectrometry (FAB-MS); X-ray data for the thymine complexes and elemental analysis for the thymidine complexes are reported. The antiproliferative activity of the complexes was tested on human chronic myelogenous leukaemia K562 cells. Arrested polymerase-chain reaction analysis was carried on to correlate antiproliferative activity and inhibition of DNA replication. All Pd and Pt complexes exhibit antiproliferative activity, Pd complexes resulting always more active than Pt complexes. Arrested PCR data are strongly in agreement with the effects on cell growth, suggesting that inhibition of the DNA replication by the synthesized compounds is the major basis for their in vitro antiproliferative activity.     

An Integrative Approach Combining Noncovalent Mass Spectrometry, Enzyme Kinetics and X-ray Crystallography to Decipher Tgt Protein-Protein and Protein-RNA Interaction

The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.     

Energetic selection of topology in ferredoxins

Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe₄S₄ metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple α+β fold is found alone or as a domain in larger proteins throughout all three kingdoms of life. Previous analyses of the heptapeptide conformation in experimentally determined ferredoxin structures revealed a pervasive right-handed topology, despite the fact that the Fe₄S₄ cluster is achiral. Conformational enumeration of a model CGGCGGC heptapeptide bound to a cubane iron-sulfur cluster indicates both left-handed and right-handed folds could exist and have comparable stabilities. However, only the natural ferredoxin topology provides a significant network of backbone-to-cluster hydrogen bonds that would stabilize the metal-peptide complex. The optimal peptide configuration (alternating α_L,α_R) is that of an α-sheet, providing an additional mechanism where oligomerization could stabilize the peptide and facilitate iron-sulfur cluster binding.     

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.     

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.     

Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.     

Studies of the interaction of potassium(I), calcium(II), magnesium(II), and copper(II) with cyclosporin A

Metal ion binding properties of the immunosuppressant drug cyclosporin A have been investigated. Complexation studies in acetonitrile solution using 1H NMR and CD spectroscopy yielded 1:1 metal-peptide binding constants (log(10)K) for potassium(I), <1, magnesium(II), 4.8+/-0.2, and calcium(II), 5.0+/-1.0. The interaction of copper(II) with cyclosporin A in methanol was investigated with UV/visible and electron paramagnetic resonance (EPR) spectroscopy. No complexation of copper(II) was observed in neutral solution. In the presence of base, monomeric copper(II) complexes were detected. These results support the possibility that cyclosporin A has ionophoric properties for biologically important essential metal ions.     

Synthesis, characterization, and antitumor activity of 1,2-bis(diphenylphosphino) ethane platinum(II) and palladium(II) complexes

A number of 1,2-bis(diphenylphosphino)ethane monomeric platinum(II) and palladium(II) complexes have been synthesized in light of their potential antitumor activity. The metal center is coordinated with a number of carboxylate anions in the cis-configuration. These complexes have been characterized by elemental analysis, conductivity measurement, and various spectroscopic techniques [IR and 195Pt NMR]. In vivo screening tests for activity of these complexes were performed against the L1210/0 murine leukemia cancer model, but none displayed a significant level of antitumor activity.     

Geometrical configuration of monomethyl–platinum(II) complexes driven by the size of entering nitrogen ligands

The reaction of the monoalkyl complex trans-[Pt(DMSO)₂Cl(CH₃)] with a large variety of heterocyclic nitrogen bases L, in chloroform solution, leads to the formation of uncharged complexes of the type [Pt(DMSO)(L)Cl(CH₃)], containing four different groups coordinated to the metal center. Only two out of the three different possible isomers were detected in solution. These two trans(C,N) and cis(C,N) species can be unambiguously identified through ¹H NMR spectroscopy. For the trans(C,N) isomers, average values of ²J_PtH=75±4 Hz and ³J_PtH=36±4 Hz have been observed for the coordinated methyl and DMSO ligands, respectively. In the case of the cis(C,N) isomers, these values increase to ²J_PtH=83±2 Hz, and decrease to ³J_PtH=26±3 Hz due to the mutual exchange of ligands in trans position to CH₃ and DMSO. In the case of bulky asymmetric ligands, such as quinoline, 2-quinolinecarboxaldehyde, 2-methylquinoline, 5-aminoquinoline, 2-phenylpyridine and 2-chloropyridine, slow rotation of the hindered group around the Pt---N bond makes the coordinated DMSO ligand prochiral. NMR experiments have shown that the first reaction product is the trans(C,N) isomer as a consequence of the very fast removal of one DMSO ligand by the nitrogen bases from the starting complex trans-[Pt(DMSO)₂Cl(CH₃)]. This trans kinetic product undergoes a geometrical conversion into the more stable cis(C,N) isomer through the intermediacy of fast exchanging aqua-species. The rate of isomerization and the relative stability of the two isomers depends essentially on the rate of aquation and on the steric congestion imposed by the new L ligand on the metal.     

113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase

Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M. (1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase. The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR. Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer. This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate. The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102. The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As) complex. Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate. The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts. Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis. The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.     

Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation

A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.     

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.     

31P NMR of alkaline phosphatase. Saturation transfer and metal-phosphorus coupling.

The rate constants which characterize the formation and breakdown of the noncovalent (E.P) and covalent (E-P) enzyme-phosphate intermediates on the alkaline phosphatase reaction pathway are known to be sensitive to the nature of the metal ion bound to the enzyme. 31P NMR saturation transfer has been demonstrated to provide a simple and sensitive method for measuring the metal ion dependence of these rates under equilibrium conditions. When the native Zn2+ was replaced by Cd2+, the 31P NMR spectrum at high pH revealed a new resonance at 12.6 ppm which has been assigned to the noncovalent enzyme.phosphate complex. Reconstituting the enzyme with enriched 113Cd2+ caused this unusually downfield-shifted resonance to appear as a doublet due to 113Cd-31P spin coupling (2J31P-O-113Cd = 30 Hz). This result provides the first unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase.     

Bioreduction of Pt (IV) from aqueous solution using sulphate-reducing bacteria

The aims of this study were to investigate the role of sulphate-reducing bacteria in facilitating Pt removal from aqueous solutions and to investigate the role of a hydrogenase enzyme in Pt reduction in vitro. To avoid precipitation of Pt as Pt sulphide, a resting (non-growing) mixed culture was used. A pH-dependent rate of Pt removal from aqueous solution was observed, indicating that metal speciation was the main factor for its removal from solution. The maximum initial concentration of Pt(IV) that the cells can effectively remove from solution was 50 mg/l, while the maximum capacity was only 4 mg of Pt per gram of resting biomass. Transmission electron microscopy and energy dispersive X-ray analyses indicated that Pt was being precipitated in the periplasm, a major area of hydrogenase activity in the cells. In vitro investigation of Pt reduction with hydrogen as the electron donor showed that 49% was removed within 1 h when a relatively pure hydrogenase extract was used, 31% was removed with a cell-free soluble extract and 70% removed by live cells.     

The photohydrochlorination of platinum(IV) chloride in chloroform

When irradiated at 240 nm, PtCl₄ in CHCl₃ is converted to H₂PtCl₆. When irradiated at wavelengths longer than 265 nm, PtCl₄ is converted to H₂PtCl₄ and H₂PtCl₆ in equal amounts. The latter reaction is suggested to proceed by dissociation of chlorine from a ligand to metal charge transfer excited state of Pt(IV) through a Pt(III) intermediate that disproportionates. The 240 nm photoreaction includes a second, solvent-initiated pathway, suggested to involve CCl₃ radicals from the photolysis of chloroform, which attack the PtCl₄ oligomer to create a Pt(V) intermediate.     

DNA recognition by metal-peptide complexes containing the recognition helix of the phage 434 repressor

 Short oligopeptides (14 residues) derived from the DNA recognition helix of the phage 434 repressor (434R) have been tethered onto the metallointercalating [Rh(phi)₂(phen′)]³⁺, and the DNA recognition characteristics of the resultant metal-peptide complexes have been examined. DNA sequence selectivities for the family of metal-peptide complexes, determined in photoactivated DNA cleavage experiments, reproduce features of operator recognition by the native 434R. Binding to the DNA duplex depends both on the appended peptide and upon the metallointercalating unit, as determined through variations in the peptide sequence and in the diastereomeric configuration of the metal-peptide. The complexes preferentially target 5′-ACAA-3′ operator sites and single-base variants, bind at 50 nM concentrations, and, as determined by chemical footprinting, protect 7–10 bp of DNA around the target sites. Comparative cleavage studies on synthetic oligonucleotides containing variations in operator sequence, furthermore, reveal a hierarchy in sequence preference which resembles the native protein, but highest affinity for the metal-peptides, unlike 434R, is found for 5′-ACGA-3′. These studies illustrate a new route to the rational design of small, artificial repressors through the construction of metal-peptide complexes. Received: 18 June 1997 / Accepted: 11 September 1997     

Evidence for hydrated spermidine-calf thymus DNA toruses organized by circumferential DNA wrapping

In spermidine-condensed calf thymus DNA preparations, torus-shaped condensates were shown by transmission electron microscopy to exist under the hydrated conditions of the freeze fracture experiment. Using extremely low Pt metal deposition levels (9 A Pt/C) high-contrast replicas of the spermidine-DNA toruses were obtained that showed circumferential wrapping of single DNA double helix-size surface fibres. Stereoscopic analysis of high magnification stereomicrographs established some details of the three-dimensional organization of two DNA double helix sections winding circumferentially on the inner surface of one such torus. These measurements demonstrate the usefulness of stereoscopic analysis of these high macromolecular organization magnification. Measurements on a number of torus-shaped complexes (n = 16) yielded these average dimensions: inner circumference (1840 +/- 204 A) outer circumference (2800 +/- 222 A), torus ring thickness (143 +/- 18 A). These data support a continuous circumferential DNA-winding model of torus organization proposed by Marx & Reynolds.