Mode of reversible binding of neocarzinostatin chromophore to DNA: base sequence dependency of binding.

The reversible binding of neocarzinostatin chromophore to polynucleotides was studied in order to understand the molecular basis of its base sequence-specificity in DNA damage production. Studies of the spectroscopic and thermodynamic properties of chromophore-polynucleotide interactions reveal that the binding of the chromophore to poly(dA-dT) is qualitatively and quantitatively different from that to poly(dG-dC) (and poly(dI-dC]. From these and other experiments using double-stranded mixtures of homopolynucleotides, it is proposed that the observed A T specific intercalation might result from differential recognition of minor variations in the B-DNA type structure by the chromophore.     

Cryospectrokinetic evidence for the mode of reversible binding of neocarzinostatin chromophore to poly(deoxyadenylic-thymidylic acid)

The spectra of neocarzinostatin (NCS) chromophore during its reversible association with poly(dA-dT).poly(dA-dT) [poly(dA-dT)] were recorded (at intervals of 17 ms or more) by a cryospectroscopic method. Examination of the spectral changes of a drug during its interaction with DNA has not been previously reported. Such studies indicate binding of chromophore to poly(dA-dT) is a two-step process in which the spectral properties of the intermediate poly(dA-dT). NCS chromophore species closely resemble those of the final equilibrium species. On the basis of cryokinetic studies (at single wavelengths) carried out at low temperature (2 degrees C), the following proposed mechanism of the DNA-drug (PD) interaction was quantitated: (Formula: see text). In analogy with the other reports on the kinetics of drug-DNA interaction, (PD)I and (PD)II could represent externally bound and intercalated complexes, respectively. However, since the spectra of (PD)I and (PD)II are closely similar, it can also be proposed that (PD)I and (PD)II represent two forms of an intercalated complex. The rate and equilibrium constant for each step were determined by examining the kinetics of the forward and reverse reactions. This was accomplished by determining the polynucleotide concentration dependence of the apparent fast and slow first-order rate constants observed during a double-exponential increase in transmittance (at 330 nm) associated with the binding and the apoprotein-induced dissociation rate constant of the chromophore from poly(dA-dT). The opportunity to use apoprotein, instead of a detergent, to follow the kinetics of the reverse reaction provides a novel approach to these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the phototoxicity of the fluorescent protein KillerRed

The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by “pre-bleached” KillerRed.     

Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.     

Flanking polyproline sequences inhibit beta-sheet structure in polyglutamine segments by inducing PPII-like helix structure

Polyglutamine (poly(Q)) expansion is associated with protein aggregation into β-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a β-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to ≈ 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with ≥ 6 glutamine residues formed β-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a “tug-of-war” between two conformations, a PPII-like helix and a β-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone β-sheet. These structural observations may shed light on the threshold phenomenon of poly(Q) aggregation, and support the hypothesized evolution of “protective” poly(P) tracts adjacent to poly(Q) aggregation domains.     

Combined enzymatic and chemical approaches to the synthesis of unique polyribonucleotides.

The enzymatic polymerization by polynucleotide phosphorylase of 6-chloro-9-(beta-D-ribofuranosyl)purine 5'-diphosphate to poly(6-chloropurinylic acid) and its conversion to poly(6-thioninosinic acid) is described. The sulfur isostere of poly(I) was found not to form a complex with poly(C), but to form a self-association complex with a Tm around 295 degrees K. The sedimentation velocities, pKa and Tm values of the polymer have been examined under various conditions. A two (or more) stranded helical array is suggested as the most probable structure. Thermal loss of the thione chromophore was noted for poly- (S6I), S6IMP and S6I; the degradation product from S6I was shown to be inosine.     

Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ_em = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λ_em = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.     

Bis(pyrrolecarboxamide) linked to intercalating chromophore oxazolopyridocarbazole (OPC): selective binding to DNA and polynucleotides.

We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator. The hybrid molecule Net-OPC binds to poly[d(A-T)] at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0). Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly[d(A-T)] than does netropsin. On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly[d(A-T)] is suggested to involve both intercalation and external binding of the OPC chromophore. In contrast, on poly[d(G-C)], Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated. The binding constant of Net-OPC to poly[d(G-C)] was found to be about 350-fold lower than that of the high-affinity binding site in poly[d(A-T)]. As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site. The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate. The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.     

Solution structure of a cyanobacterial phytochrome GAF domain in the red-light-absorbing ground state

The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B′ cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80° relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B′ GAF domain can, by itself, complete the Pr → Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.     

Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K_a versus [Na⁺] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp^o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were − 98, − 140, − 120 and − 110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

Chromophore Packing Leads to Hysteresis in GFP

Green fluorescent protein (GFP) possesses a unique folding landscape with a dual basin leading to the hysteretic folding behavior observed in experiment. While theoretical data do not have the resolution necessary to observe details of the chromophore during refolding, experimental results point to the chromophore as the cause of the observed hysteresis. With the use of NMR spectroscopy, which probes at the level of the individual residue, the hysteretic intermediate state is further characterized in the context of the loosely folded isomerized native-like state {N_iso} predicted in simulation. In the present study, several residues located in the lid of GFP indicate heterogeneity of the native states. Some of these residues show chemical shifts when the native-like intermediate {N_iso} responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in {N_iso} as predicted in recent theoretical work. Here, we observed that multiple chromophore environments within the native state are averaged in the trapped intermediate, linking chromophore flexibility to mispacking in the trapped intermediate. The present work is experimental evidence for the proposed final “locking” mechanism in GFP folding forming an incorrectly or loosely packed barrel under intermediate (hysteretic) folding conditions.     

Neocarzinostatin chromophore-DNA adducts: evidence for a covalent linkage to the oxidized C-5'' of deoxyribose.

The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Study by circular dichroism and optical rotatory dispersion of polypeptides with aromatic side-chain chromophores

Poly(ortho-, meta-, and para-γ-nitrobenzyl-L -glutamates) were studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in two helicogenic solvents, hexafluoroisopropanol (HFIP) and dichloroethane (EDC), and two non-helicogenic solvents, dichloracetic acid (DCA) and trifluoroacetic acid (TFA). The corresponding glutamates were also studied in DCA and TFA. The symmetric nitrobenzylic chromophore is optically active when the polymers are in solution in DCA and TFA. The corresponding glutamates are also optically active under the same conditions. Thus, it was not possible to explain the origin of the optical activity of the side-chain chromophore when the polymer is in solution in a helicogenic solvent. Nevertheless, from a side-chain dichroic band, a helix–coil transition curve was determined and the stability of each poly(γ-nitrobenzyl-L -glutamate) given; this stability depends on the position of the nitro substituent on the aromatic ring.     

Competition between anaerobic covalent linkage of neocarzinostatin chromophore to deoxyribose in DNA and oxygen-dependent strand breakage and base release

Treatment of poly(dA-dT) X poly(dA-dT) with the nonprotein chromophore of neocarzinostatin in the presence of sulfhydryls resulted in both direct and alkali-dependent base release, indicative of DNA sugar oxidation. Covalent chromophore-DNA adducts were also formed. Under anaerobic conditions, base release was strongly inhibited; however, adduct formation was not inhibited and in some cases was markedly enhanced. In the presence of dithiothreitol, anoxia increased adduct formation by a factor of 2, and a particularly stable adduct species was formed, which was recovered from nuclease digests of the treated DNA as a highly fluorescent compound with structure chromophore-d(TpApT). Acid hydrolysis of chromophore-d(TpApT) released free adenine base and both 3'dTMP and 5'dTMP, leaving a compound that contained only chromophore and the deoxyadenosine sugar. These results conclusively confirm that the chromophore forms a covalent adduct with deoxyribose in DNA. Thus, even in the absence of oxygen, activation of the chromophore by sulfhydryls results in the formation of a species capable of reacting with deoxyribose. Several other adduct species were also formed, some of which were nonfluorescent and relatively hydrophilic, but all of which were produced in increased amounts under anoxia. This inverse relation between sugar oxidation and adduct formation suggests that the two lesions share a common precursor. In the presence of other thiols, the effects of anoxia were somewhat different. With glutathione, anoxia markedly enhanced adduct formation, but the total adduct formed was considerably less than with dithiothreitol. With 2-mercaptoethanol, anoxia had no effect on total adduct formation, but the distribution of adduct species was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulation of poly(spiropyran-L-glutamate): Influence of chromophore isomerization

A study of the influence of the spiropyran to merocyanine ring opening on a model of poly(spiropyran-L -glutamate) as implied by the experimental data (T. M. Cooper, K. A. Obermeier, L. V. Natarajan, and R. L. Crane (1992) Photochemistry and Photobiology, 55, 1–7) is presented. The individual chromophore is studied by the AM1 semiempirical approach, while molecular mechanics and dynamics calculations are employed in the analysis of the poly(spiropyran-L -glutamate) model. It is shown that the α-helical secondary structure is less conserved in the polypeptide substituted with the merocyanine form of the chromophore. In particular, larger side-chain flexibility, increased backbone hydrogen-bond lengths, as well as a larger helix bending are calculated. Furthermore, a random conformational minimization calculation finds the intrinsic behavior of the spiropyran molecular system as being more of a helix “maker” than its merocyanine analogue. The interactions of the chromophore substituent with other side chains prove, in part, that an early event in the decay of the α-helical structure is the formation of hydrogen bonds between the carboxylic acid groups and the merocyanine oxygens. The results lend support to the experimental observation that the merocyanine group destabilizes the α-helical framework of the polypeptide, thus possibly allowing the entry of solvent molecules into the α-helical core, while spiropyran in its closed form shields it from the solvent. © 1992 John Wiley & Sons, Inc.     

A structural basis for the pH-dependent increase in fluorescence efficiency of chromoproteins

Within the fluorescent protein and chromoprotein family, the phenomenon of photoswitching is both intriguing and biotechnologically useful. Illumination of particular chromoproteins with intense light results in dramatic increases in fluorescence efficiency (termed kindling) and involves cis-trans isomerization of the chromophore. Here we report that chromophore isomerization can also be driven via alteration in pH. Specifically, we demonstrate that a number of naturally occurring chromoproteins, and their engineered variants, undergo a dramatic 20-100-fold increase in fluorescence efficiency at alkaline pH (>pH9.0). We have determined to 1.8 A resolution the structure of one such chromoprotein, Rtms5(H146S), in its highly far-red fluorescent form (Phi(F), 0.11 at pH 10.7) and compared it to the structure of the non-fluorescent form (Phi(F), 0.002 at pH 8.0). At high pH, the cyclic tri-peptide chromophore was observed to be mobile and distributed between a trans non-coplanar and a cis coplanar conformation, whereas at the lower pH, only a trans non-coplanar chromophore was observed. Calculation of pK(a) values suggested that titration of the side-chain of the conserved Glu215 close to the chromophore is involved in promoting the cis-coplanar conformation. Collectively, our data establish that isomerization to form a coplanar chromophore is a basis of the increased fluorescence efficiency at high pH. The phenomenon of pH-induced fluorescence gain has similarities with photoswitching, thereby providing a model to study the mechanism of kindling.     

Molecular and evolutionary aspects of microbial sensory rhodopsins

Retinal proteins (~ rhodopsins) are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices which bind the chromophore retinal (vitamin A aldehyde). They are widely distributed through all three biological kingdoms, eukarya, bacteria and archaea, indicating the biological significance of the retinal proteins. Light absorption by the retinal proteins triggers a photoisomerization of the chromophore, leading to the biological function, light-energy conversion or light-signal transduction. This article reviews molecular and evolutionary aspects of the light-signal transduction by microbial sensory receptors and their related proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.     

The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.