Actinomycin D binds strongly to d(CGACGACG) and d(CGTCGTCG)

Earlier calorimetric studies had indicated that despite the absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds strongly to actinomycin D (ACTD) with high cooperativity and a 2:1 drug/duplex ratio. A subsequent optical spectral study with related oligomers led us to suggest that ACTD may likely stack at the G. C basepairs of the duplex termini. New findings are reported herein to indicate that despite the lack of complete self-complementarity, oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD affinities with binding constants of roughly 2 x 10(7) M(-1) and binding densities of 1 drug molecule per strand. The ACTD binding affinity for the corresponding heteroduplex obtained by annealing these two oligomers is, however, considerably reduced. Although spectroscopic results with related oligomers obtained by removing, replacing, or appending bases at the termini appear to be consistent with the end-stacking model, capillary electrophoretic (CE) evidence provides additional insights into the binding mode. CE experiments with the self-complementary oligomers d(CGAGCTCG) and d(CGTCGACG) revealed contrasting migration patterns in the presence of ACTD, with mobility retardation and acceleration exhibited by the GpC- and non-GpC-containing octamers, respectively, whereas the X/X-mismatched d(CGXCGXCG) experienced retardation. These results, along with those of related oligomers, suggest that ACTD may in fact stack at the duplex stem end of a monomeric hairpin or at the 3'-end of dG as a single strand. The seemingly cooperative ACTD binding and the curved Scatchard plot for the self-complementary d(CGTCGACG) may thus be attributed to the drug-induced duplex denaturation resulting from strong binding to single strands of d(CGXCGYCG) motif. Detailed structural information on the ACTD-DNA complexes, however, must await further NMR investigations.     

Actinomycin D binds strongly to d(TGTCATTG), a single-stranded DNA devoid of GpC sites

Despite the absence of the GpC sequence and complete self-complementarity, d(CGTCGTCG) has recently been shown to bind strongly to actinomycin D (ACTD) with a binding density of about one drug molecule per strand. To further elucidate the nature of such a binding, studies are herein made with single-base G --> A and C --> T replacements in d(CGTCGTCG) to identify the DNA bases that play important roles in the strong ACTD binding of this oligomer. On the basis of these results, the octamer d(TGTCATTG) has been identified as a potentially strong ACTD binder. Indeed, binding titration confirms such an expectation and reveals an ACTD binding constant of about 1 x 10(7) M(-1) and a binding density of roughly 0.8 drug molecule per DNA strand for this strong binding mode. Similar binding studies with single-base substitutions on d(TGTCATTG) further reveal the relative importance of the C and G bases on its ACTD binding, with the 3'-terminus G appearing to be the most crucial base. Further base substitutions lead to the conclusion that these C and G bases act in concert rather than individually in the ACTD binding of d(TGTCATTG). Spectral comparisons with the apparently single-stranded GpC-containing d(TGCTTTG) led to the proposal of a speculated monomeric hairpin binding model to account for the experimental observations. This model makes use of the notion that ACTD prefers to have the 3'-sides of both G bases stacking on the opposite faces of its planar phenoxazone chromophore, a principle akin to its classic preference for the GpC sequence in duplex form. The finding that ACTD can bind strongly to single-stranded DNA of special sequence motifs may have important implications.     

Actinomycin D in low concentrations binds uniformly to human chromosomes

The binding of actinomycin D (actD) to fixed human metaphase chromosomes was studied by using autoradiography with [3H]actD and indirect immunofluorescence with a specific anti-actD antibody. At concentrations of 0.01 and 0.1 micrograms/ml there was a uniform distribution of drug along the chromosomes as observed by both methods. This is the first study to date characterizing actD binding at such low concentrations to human chromosomes. Since actD intercalates into the DNA helix with GC specificity, our observations indicate that detectable differences in base composition along the lengths of human chromosomes are minimal.     

A Combined 2D-NMR and Molecular Dynamics Analysis of the Structure of the Actinomycin D: d(ATGCAT)2 Complex

Abstract

We present a comparative analysis of an NMR experiment and molecular and harmonic dynamics simulations of an actinomycin D: d(ATGCAT)₂ complex. A comparison of NOE measurements and 1/R⁶ weighted proton-proton distances confirm the general correctness of the Actinomycin D-DNA model proposed by Sobell. There are, however, some substantial differences between the proton-proton distances inferred from the NOE results and the molecular and harmonic dynamics simulations. The remaining discrepancies could either come from contributions of other conformations to the average properties of the complex or from uncertainties in the NMR distance analysis. An analysis of the molecular dynamics helix properties, sugar puckers, hydrogen bonding, rms fluctuations and torsional properties are qualitatively consistent with those from previous simulations, but the presence of an intercalated drug leads to some new structural and dynamical features.     

Interleukin-2 binds to ganglioside GD(1b)

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.     

Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D.

The crystal structures of the 2:1 complex of the self-complementary DNA octamer d(GAAGCTTC) with actinomycin D has been determined at 3.0 A resolution. This is the first example of a crystal structure of a DNA-drug complex in which the drug intercalates into the middle of a relatively long DNA segment. The results finally confirmed the DNA-actinomycin intercalation model proposed by Sobell & co-workers in 1971. The DNA molecule adopts a severely distorted and slightly kinked B-DNA-like structure with an actinomycin D molecule intercalated in the middle sequence, GC. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base-peptide and chromophore-backbone hydrogen bonds. The DNA helix appears to be unwound by rotating one of the base-pairs at the intercalation site. This single base-pair unwinding motion generates a unique asymmetrically wound helix at the binding site of the drug, i.e. the helix is loosened at one end of the intercalation site and tightened at the other end. The large unwinding of the DNA by the drug intercalation is absorbed mostly in a few residues adjacent to the intercalation site. The asymmetrical twist of the DNA helix, the overall conformation of the two cyclic depsipeptides and their interaction mode with DNA are correlated to each other and rationally explained.     

Unique actinomycin D binding to self-complementary d(CXYGGCCY'X'G) sequences: duplex disruption and binding to a nominally base-paired hairpin

Actinomycin D (ACTD) has been shown to bind weakly to the sequence -GGCC-, despite the presence of a GpC site. It was subsequently found, however, that d(CATGGCCATG) binds relatively well to ACTD but exhibits unusually slow association kinetics, contrary to the strong-binding -XGCY- sites. In an effort to elucidate the nature of such binding and to delineate the origin of its interesting kinetic behavior, studies have now been extended to include oligomers with the general sequence motifs of d(CXYGGCCY′X′G)₂. It was found that analogous binding characteristics are observed for these self-duplex decamers and comparative studies with progressively base-truncated oligomers from the 5′-end led to the finding that d(GGCCY′X′G) oligomers bind ACTD considerably stronger than their parent decamers and exhibit 1:1 drug/strand binding stoichiometry. Melting profiles monitored at the drug spectral region indicated additional drug binding prior to the onset of eventual complex disruptions with near identical melting temperatures for all the oligomers studied. These results are consistent with the notion that the related oligomers share a common strong binding mode of a hairpin-type, with the 3′-terminus G folding back to base-pair with the C base of GGC. A binding scheme is proposed in which the oligomers d(CXYGGCCY′X′G) exist predominantly in the duplex form and bind ACTD initially at the central GGCC weak site but subsequently disrupt to accommodate the stronger hairpin binding and thus the slow association kinetics. Such a mechanism is supported by the observation of distinct biphasic fluorescence kinetic traces in the binding of 7-amino-ACTD to these duplexes.     

On the stoichiometry of Deinococcus radiodurans Dps-1 binding to duplex DNA

DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans, which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core. Composed of six Dps-1 dimers, each capable of DNA binding by N-terminal extensions interacting in consecutive DNA major grooves, dodecameric Dps-1 would be predicted to feature six DNA binding sites. Using electrophoretic mobility shift assays and intrinsic tryptophan fluorescence, we show that dodecameric Dps-1 binds 22-bp DNA with a stoichiometry of 1:6, consistent with the existence of six DNA binding sites. The stoichiometry of Dps-1 binding to 26-bp DNA is 1:4, suggesting that two Dps-1 dodecamers can simultaneously occupy opposite faces of this DNA. Mutagenesis of an arginine (Arg132) on the surface of Dps-1 leads to a reduction in DNA binding. Altogether, our data suggest that duplex DNA lies along the dimer interface, interacting with Arg132 and the N-terminal α-helices, and they extend the hexagonal packing model for Dps-DNA assemblies by specifying the basis for occupancy of available DNA binding sites.     

Structural analysis of the decanucleotide duplex d(GGTAATTACC)2 using 1H NMR spectroscopy.

The conformation of the decanucleotide duplex d(GGTAATTACC)2 has been investigated in solution by one- and two-dimensional proton NMR spectroscopy. Intra- and inter-nucleotide two-dimensional nuclear Overhauser enhancement data, recorded at mixing times between 15 and 250 ms, reveal a right-handed B-DNA structure. The data also show that the A-T basepairs of the TAATTA tract are highly propeller twisted and the minor groove is particularly narrow.     

Conformational properties and thermodynamics of the RNA duplex r(CGCAAAUUUGCG)2: comparison with the DNA analogue d(CGCAAATTTGCG)2.

The thermodynamic stability of nine dodecamers (four DNA and five RNA) of the same base composition has been compared by UV-melting. TheDeltaG of stabilisation were in the order: r(GACUGAUCAGUC)2>r(CGCAAATTTGCG)2 approximately r(CGCAUAUAUGCG)2>d(CGCAAATTTGCG)2 approximately r(CGCAAAUUUGCG)2>d(CGCATATATGCG)2 approximately d(GACTGATCAGTC)2>r(CGCUUUAAAGCG)2 approximately d(CGCTTTAAAGCG)2. Compared with the mixed sequences, both r(AAAUUU) and r(UUUAAA) are greatly destablising in RNA, whereas in DNA, d(TTTAAA) is destabilising but d(AAATTT) is stabilising, which has been attributed to the formation of a special B'structure involving large propeller twists of the A-T base pairs. The solution structure of the RNA dodecamer r(CGCAAAUUUGCG)2has been determined using NMR and restrained molecular dynamics calculations to assess the conformational reasons for its stability in comparison with d(CGCAAATTTGCG)2. The structures refined to a mean pairwise r.m.s.d. of 0.89+/-0.29 A. The nucleotide conformations are typical of the A family of structures. However, although the helix axis displacement is approximately 4.6 A into the major groove, the rise (3.0 A) and base inclination ( approximately 6 degrees ) are different from standard A form RNA. The extensive base-stacking found in the AAATTT tract of the DNA homologue that is largely responsible for the higher thermodynamic stability of the DNA duplex is reduced in the RNA structure, which may account for its low relative stability.     

d(TTTCCTCGCCGGAAA)的一维NMR氢谱分析

单链d(TTTCCTCGCCGGAAA)易溶于水,且由于其本身存在序列特异性,即可形成分子内“发夹”结构,本实验分别测得其在全重水(D2O)、92?O 8%H2O溶液中的一维1H谱,认为环出区域碱基质子的共振峰与其他同种质子的共振峰有明显的区别,主要表现在其共振峰会明显移向高场区。     

Interaction of Lambda- and Delta-[Ru(bpy)2(pbmz)](PF6)2 with the oligonucleotide duplex d(CGCGAATTCGCG)2

The interaction of the enantiomeric complexes Lambda- and Delta-[Ru(bpy)(2)(pbmz)](PF(6))(2) (bpy=2,2'-bipyridine, pbmz=2-(2'-pyridyl)benzimidazole) with the DNA duplex d(CGCGAATTCGCG)(2) was investigated by means of 2D NMR techniques. The synthesis of the enantiomers was based on the optically pure complexes Lambda- and Delta-[Ru(bpy)(2)(py)(2)](2+) and were characterized by CD and NMR spectroscopy. NMR data indicate that both enantiomers bind weakly to the oligonucleotide, approaching from the minor groove at the centre of the helix. The perturbation of the B-DNA conformation is minor with an apparent absence of enantioselectivity. Molecular modelling calculations in conjunction with the NOE data support the suggestion that more than one binding modes are present. The imidazole amine group of the pbmz ligand is probably hydrogen bonded to the DNA phosphodiesteric backbone at the AATT step, and this may provide an explanation for the diminished enantioselectivity observed.     

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2

Summary Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)₂, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraints is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of ¹H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.Abbreviations DNA deoxyribonucleic acid - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy     

Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth

Calmyrin1 (CaMy1) is an EF-hand Ca²⁺-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca²⁺-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca²⁺ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

1H-NMR studies of a monointercalating drug into a d(CpGpApTpCpG)2 minihelix

The structure of the complex formed between the 7H-pyridocarbazole monomer [[(2-piperidyl)-2,1-ethane-yl] [10-methoxy-7H-pyrido[4,3-c]carbazolium] dimethane sulfonate] and the autocomplementary hexanucleotide d(CpGpApTpCpG)2 in aqueous solution is analyzed by 270- and 400-MHz 1H-nmr. The large upfield shifts observed for both the drug and the self-complementary hexanucleotide protons provide evidence for intercalated complexes. The observation of intermolecular nuclear Overhauser effects between drug and the hexanucleotide protons gives a privileged orientation of the drug in the intercalation site with the quaternarizing ethyl piperidine chain protruding in the major groove. Moreover, the data suggest an intercalation based on the neighbor exclusion site principle in the three alternating sequences.     

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Structure and dynamics of distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at low drug:DNA ratios

Two-dimensional NMR has been used to study the interaction of distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at low and intermediate drug:DNA ratios (less than 2.0). Drug-DNA contacts were identified by nuclear Overhauser effect spectroscopy, which also served to monitor exchange of the drug between different binding sites. At low drug:DNA ratios (0.5), distamycin A binds in two orientations within the five central A-T base pairs and has a preference (2.2:1) for binding with the formyl end directed toward the 5' side of the A-rich strand. The pattern of drug-DNA contacts corresponding to the preferred binding orientation are consistent with the drug sliding between adjacent AAAT and AATT binding sites at a rate that is fast on the NMR time scale. Similarly, the pattern of NOEs associated with the less favored orientation are consistent with the drug sliding between adjacent AATT and ATTT sites, again in fast exchange. Off-rates for the drug from the major and minor binding orientations were measured to be 2.4 +/- 1.5 and 3.3 +/- 1.5 s-1, respectively, at 35 degrees C. At intermediate drug:DNA ratios (1.3) exchange of the drug between the two one-drug and the two sites of a two-drug complex is observed. Off-rates for both drugs from the 2:1 complex were measured to be 1.0 +/- 0.5 s-1 (35 degrees C).     

Sequence-dependent effects in drug-DNA interaction: the crystal structure of Hoechst 33258 bound to the d(CGCAAATTTGCG)2 duplex.

The bis-benzimidazole drug Hoechst 33258 has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The structure has been solved by molecular replacement and refined to an R factor of 18.5% for 2125 reflections collected on a Xentronics area detector. The drug is bound in the minor groove, at the five base-pair site 5'-ATTTG and is in a unique orientation. This is displaced by one base pair in the 5' direction compared to previously-determined structures of this drug with the sequence d(CGCGAATTCGCG)2. Reasons for this difference in behaviour are discussed in terms of several sequence-dependent structural features of the DNA, with particular reference to differences in propeller twist and minor-groove width.