Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix

The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.     

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.     

Conformational and model-building studies of the hairpin form of the mismatched DNA octamer d(m5C-G-m5C-G-T-G-m5C-G)

The hairpin form of the mismatched octamer d(m5C-G-m5C-G-T-G-m5C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3'-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5'-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5'-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual gamma' and beta+ torsion angles in residue dG(6). The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone. The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T-A-T4-T-A-G-G-A-T), in which the central -T4- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5''-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.     

Conformational flexibility of N-glycans in solution studied by REMD simulations

Protein–glycan recognition regulates a wide range of biological and pathogenic processes. Conformational diversity of glycans in solution is apparently incompatible with specific binding to their receptor proteins. One possibility is that among the different conformational states of a glycan, only one conformer is utilized for specific binding to a protein. However, the labile nature of glycans makes characterizing their conformational states a challenging issue. All-atom molecular dynamics (MD) simulations provide the atomic details of glycan structures in solution, but fairly extensive sampling is required for simulating the transitions between rotameric states. This difficulty limits application of conventional MD simulations to small fragments like di- and tri-saccharides. Replica-exchange molecular dynamics (REMD) simulation, with extensive sampling of structures in solution, provides a valuable way to identify a family of glycan conformers. This article reviews recent REMD simulations of glycans carried out by us or other research groups and provides new insights into the conformational equilibria of N-glycans and their alteration by chemical modification. We also emphasize the importance of statistical averaging over the multiple conformers of glycans for comparing simulation results with experimental observables. The results support the concept of “conformer selection” in protein–glycan recognition.     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Poly[d(A-T)-Cs+] conformations studied by IR spectroscopy

Infrared absorption and ir linear dichroism measurements have been performed on poly[d(A-T)-Cs⁺] films at various relative humidities. At high relative humidity, samples are in a B form; at low relative humidity, in a C form. The B → C conformational transition is shown to be a noncooperative one corresponding to a gradual evolution of the backbone geometry of the polynucleotide within the B family. the C-form-type spectrum is characteristic of an alternated phosphodiester chain with a dinucleotidic repeat unit.     

Conformational analysis of r(CGCGCG) in aqueous solution: an A-type double helical conformation studied by two-dimensional nuclear Overhauser effect spectroscopy.

The conformation of the hexanucleoside pentaphosphate r( CGCGCG ) in aqueous solution was studied by circular dichroism, 1H- and 31P-NMR spectroscopy. The base-, H1'- and H2'-proton resonances were assigned by means of 2D-NOE spectroscopy. The base- and H1'-proton chemical shifts were studied as a function of temperature. Proton-proton distances are computed in A- and A'-RNA as well as in A-, B- and Z-DNA. A qualitative interpretation of the observed 2D-NOE intensities shows that r( CGCGCG ) adopts a regular A-type double helical conformation under our experimental conditions. The CD- and 31P-NMR experiments described in this paper are in agreement with this structure both under low- and high-salt conditions.     

The B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz two-dimensional NMR study

The non-exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C-G)3 and d(br5C-G)3 in the B form as well as in the Z form were assigned by means of two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non-exchangeable proton resonances of oligonucleotides in the Z form.     

Conformational behavior of poly-N5-(3-hydroxypropyl)-L-glutamine in water-methanol mixtures studied by 13C Nmr and CD spectroscopy

The helix–coil transition of poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPLG) has been studied in methanol–water by CD and cmr spectroscopy. For polydisperse PHPLG, two separate peaks arising from residues in helical and random-coil conformations are observed during the transition for both main-chain carbons. These results are discussed and compared to those observed in the case of a polymer sample obtained by racemization of PHPLG in 0.1 M NaOH and of PHPLG samples of controlled molecular weight and dispersity. The dominant influence of the molecular-weight heterogeneity on the double-peak phenomenon has been verified. The linewidths and chemical shift of the ¹³C resonances are discussed in terms of side-chain–main-chain interactions and side-chain solvation.     

Z-DNA's conformer substates revealed by FT-IR difference spectroscopy of nonoriented left-handed double helical poly(dG-dC)

Nonoriented hydrated films of double helical poly(dG-dC) in the Z-form were studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated slow-cooled samples between 290 and 220 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200, 220, and 240 K. IR spectral changes on isothermal relaxation at 200 and 220 K toward equilibrium, caused by interconversion of two conformer substates (CS) called Z1 and Z2, are revealed by IR difference spectra. Pronounced spectral changes on Z1-to-Z2 interconversion occur between approximately 750-1250 cm(-1) and these are attributed to structural changes of the phosphodiester-sugar backbone caused by changes of torsion angles, and to decreasing hydrogen-bonding of the ionic phosphate group with water molecules. These spectral changes on Z1-to-Z2 transition can be related to structural differences between ZI and ZII CS observed in single crystals. ZI/ZII CS occurs only at (dGpdC) base steps, and similar behavior is assumed for Z1/Z2. The Z1/Z2 population ratio was determined via curve resolution of marker bands for Z1 and Z2 centered at 785 and 779 cm(-1). This ratio is 0.64 at 290 K, corresponding to 39% of the phosphates of the (dGpdC) base steps in Z1 and 61% in Z2, and it increases to 1.24 on cooling to 220 K. For the Z2<=>Z1 equilibrium, an enthalpy change of -4.9 +/- 0.2 kJ mol(dGpdC)(-1) is obtained from the temperature dependence of the equilibrium constant. Z1 interconverts into Z2 at isothermal relaxation at 200 and 220 K, whereas on slow cooling from ambient temperature, Z2 interconverts into Z1. This unexpected reversal of CS interconversion is attributed to slow restructuring of hydration shells of the CS on quenching, in the same manner reported by Pichler et al. for the BI and BII CS of B-DNA (J. Phys. Chem. B 106, 3263-3274 (2002)). IR difference curves demonstrate two time scales on isothermal relaxation of Z1-->Z2 interconversion, a fast one for structural relaxation of the sugar-phosphate backbone, and a slow one for relaxation of the hydration shells. This slowing down of restructuring of CS hydration shells at approximately 220-240 K could be the cause for the suppression of biological functions at low temperatures.     

Right-handed and left-handed helices of poly(dA-dC) X (dG-dT).

The secondary structures of poly(dA-dC) X (dG-dT) were studied using CD and IR spectroscopies. We give spectroscopic evidence of secondary structure transitions of poly(dA-dC) X (dG-dT) from a B to a Z-like helix, induced by transition metal ions (Ni2+) in presence of high concentrations of Cs+ and Na+. In the presence of Na+, the B in equilibrium Z transition occurs at any temperature, whereas premelting conditions are required in presence of Cs+. For these two alkali ions the Z-like form is only induced by Ni2+ ions through their specific interactions at N7 of purines, under conditions of low water activity due to the high alkali salt concentration. We also show that the CD spectrum obtained in presence of Cs+ ions and characterized by a negative band at 275 nm, cannot be interpreted in terms of Z-like left-handed helix but reflects a modified B right-handed helix.     

The B----Z transition in two synthetic oligonucleotides: d(C-2-amino-ACGTG) and d(m5CGCAm5CGTGCG) studied by IR, NMR and CD spectroscopies.

The sequences CA'CGTG (where A' = 2-aminodeoxyadenosine) and m5CGCAm5CGTGCG are prepared and studied by IR, CD and 1H-NMR. Infrared spectra demonstrate the capacity of the modified hexamer and decamer to adopt a Z conformation. The influence of the NH2 substitution on the adenine or of the methylated terminal part of the decamer acting with the increase of the DNA concentration stabilizes the Z conformation at room temperature in low humidity films. Very weak proportion of Z conformation is detected in UV dilute solutions. In more concentrated NMR solutions, the Z proportion induced by high salt content is only 20-25%. The effects of the concentration and of the covalent modification of the bases are discussed.     

Conformational transitions and aggregation in poly(dA)-poly(dT) system induced by Na+ and Mg2+ ions

Effects of Mg²⁺ ions on thermally induced conformational transitions in the synthetic poly(dA)·poly(dT) and poly(dA)·2poly(dT) were studied in the buffered solutions (pH 6.9), containing 0.1 or 1 M NaCl at polynucleotide concentration of 0.1–0.3 mM (in nucleic bases). The experiments consist of measurements of the UV absorption and intensity of conventional visible static light scattering. The diagram of conformational transitions in the poly(dA)–poly(dT)–Mg²⁺ system was constructed on a basis of experimental data obtained. Anomalously strong light scattering, like critical opalescence, has been revealed at 0.1 M NaCl and [Mg²⁺]≥20 mM in the melting range of both polynucleotides, which eventually disappeared after the completion of polymer strands separation. The effect presumably is caused by a fluctuation process of polymer strands complexing which arises at a certain concentration of Mg²⁺ ions.     

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.     

An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C-G-m5C-G-A-G-m5C-G) in solution. The B-duplex and hairpin forms

By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.     

Conformational changes of purified (Na+ + K+)-ATPase detected by a sulfhydryl fluorescence probe.

The fluorescent sulfhydryl reagent S-mercuric-N-dansyl cysteine (Dn-Cys-Hg+) has been used to label a purified preparation of the (Na+ + K+)-ATPase obtained from the electric organ of Electrophorous electricus. The labelled (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), although reversibly inhibited, was capable of undergoing conformational changes associated with the active enzyme that could be monitored fluorometrically. The presence of ligands (Na+ + Mg2+ + ATP or Mg2+ + Pi) which are known to convert the enzyme from the E-1 state to the E-2-P state brought about large (97--100%) increases in fluorescence of the dimethylaminonaphthalene sulfonyl (Dn) label. An E-2 state could be achieved by the addition of Mg2+ which caused only a 32.3% increase in fluorescence over the E-1 state. Neither AMP nor TTP with or without Mg2+ or Na+ or Pi added without Mg2+ had any effect on the Dn fluorescence. If the enzyme was denatured, no fluorescence changes were observed. Small changes in the polarization of fluorescence of the Dn moiety were observed under all the conditions used. These small polarization changes and the large increases in the fluorescence intensity suggest that the enzyme can change conformational states in the presence of appropriate ligands and these conformational changes may take place in a relatively limited region of the protein's structure.     

Ethidium binding to left-handed (Z) DNAs results in regions of right-handed DNA at the intercalation site

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.     

(CGA)(4): parallel, anti-parallel, right-handed and left-handed homoduplexes of a trinucleotide repeat DNA.

Conformational properties of microsatellite DNA regions are the probable reason of their expansions in genomes which lead to serious genetic diseases in some cases. Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we study in this paper conformational properties of (CGA)(4) and compare them with those of (CAG)(4) - a related repeat, connected with Huntington's disease. We show that (CGA)(4) can adopt several distinct conformations in solution. Around neutral pH it forms a parallel-stranded homoduplex containing C(+).C, G.G, and A.A base pairs. Under the same conditions (CAG)(4) forms a hairpin. At slightly alkaline pH values and low ionic strength, (CGA)(4) also folded into a hairpin which transformed into a bimolecular anti-parallel homoduplex at increasing salt concentrations. The duplex easily isomerized into left-handed Z-DNA, implying that the mismatched adenines between G.C pairs facilitate rather than hinder the B-Z transition. No similar changes took place with (CAG)(4). Thus, the conformational repertoire of (CGA)(4) includes parallel, anti-parallel, right-handed, and left-handed homoduplexes. In contrast, (CAG)(4) invariably adopts only a single conformation, namely the very stable hairpin.