Effect of Coordinated Ions on Structure and Flexibility of Parallel G-quadruplexes: A Molecular Dynamics Study

Abstract

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G₇) quadruplex structures with Na⁺ or K⁺ ions coordinated in the cavity formed by the O6 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 Å from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na⁺ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, O6 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na⁺ ions, within the quadruplex cavity, are more mobile than coordinated K⁺ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

Effect of Coordinated Ions on Structure and Flexibility of Parallel G-quadruplexes: A Molecular Dynamics Study

Abstract Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G(7)) quadruplex structures with Na(+) or K(+) ions coordinated in the cavity formed by the O6 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 ? from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na(+) counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, O6 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na(+) ions, within the quadruplex cavity, are more mobile than coordinated K(+) ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

Effect of coordinated ions on structure and flexiblity of parallel G-quandruplexes: a molecular dynamics study

Single tract guanine residues can associate to form stable parallel quadruplex structures in the presence of certain cations. Nanosecond scale molecular dynamics simulations have been performed on fully solvated fibre model of parallel d(G7) quadruplex structures with Na+ or K+ ions coordinated in the cavity formed by the 06 atoms of the guanine bases. The AMBER 4.1 force field and Particle Mesh Ewald technique for electrostatic interactions have been used in all simulations. These quadruplex structures are stable during the simulation, with the middle four base tetrads showing root mean square deviation values between 0.5 to 0.8 A from the initial structure as well the high resolution crystal structure. Even in the absence of any coordinated ion in the initial structure, the G-quadruplex structure remains intact throughout the simulation. During the 1.1 ns MD simulation, one Na+ counter ion from the solvent as well as several water molecules enter the central cavity to occupy the empty coordination sites within the parallel quadruplex and help stabilize the structure. Hydrogen bonding pattern depends on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate cations of different sizes. In the absence of any coordinated ion, due to strong mutual repulsion, 06 atoms within G-tetrad are forced farther apart from each other, which leads to a considerably different hydrogen bonding scheme within the G-tetrads and very favourable interaction energy between the guanine bases constituting a G-tetrad. However, a coordinated ion between G-tetrads provides extra stacking energy for the G-tetrads and makes the quadruplex structure more rigid. Na+ ions, within the quadruplex cavity, are more mobile than coordinated K+ ions. A number of hydrogen bonded water molecules are observed within the grooves of all quadruplex structures.     

A COMPARISON OF THE PROPERTIES AND THE SOLUTION STRUCTURE FOR RNA AND DNA QUADRUPLEXES WHICH CONTAIN TWO GGAGG SEQUENCES JOINED WITH A TETRANUCLEOTIDE LINKER

Abstract

We have determined solution structure of r(GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. In order to obtain insight into the difference between RNA and DNA quadruplexes, we synthesized the corresponding DNA 14-mer, d(GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. K⁺ ions increased the thermal stability of both R14 and D14 structures. The binding affinity of K⁺ ions to R14 was much higher than that to D14. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K⁺ ions; two molecules of D14 form a quadruplex with both antiparallel and parallel strand alignments and with diagonal loops at both ends of the stacked G-tetrads. The NMR study also gave results that are consistent with such structure: alternate glycosidic conformation, 5′G(syn)-G(anti)3′, and characteristic chemical shift data observed for many quadruplexes containing diagonal TTTT loops.     

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G₃(TTAG₃)₃ forms an antiparallel quadruplex of the same basket type in solution containing either K⁺ or Na⁺ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K⁺-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G₃(TTAG₃)₃ motif. Both G₃(TTAG₃)₃ and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K⁺-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K⁺-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG₃(TTAG₃)₃ by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K⁺ ions.     

The formation pathway of tetramolecular G-quadruplexes

Oligonucleotides containing guanosine stretches associate into tetrameric structures stabilized by monovalent ions. In order to describe the sequence of reactions leading to association of four identical strands, we measured by NMR the formation and dissociation rates of (TGnT)₄ quadruplexes (n = 3–6), their dissociation constants and the reaction orders for quadruplex formation. The quadruplex formation rates increase with the salt concentration but weakly depend on the nature (K⁺, Na⁺ or Li⁺) of the counter ions. The activation energies for quadruplex formation are negative. The quadruplex lifetimes strongly increase with the G-tract length and are much more longer in K⁺ solution than in Na⁺ or Li⁺ solutions. The reaction order for quadruplex formation is 3 in 0.125 M KCl and 4 in LiCl solutions. The kinetics measurements suggest that quadruplex formation proceeds step by step via sequential strand association into duplex and triplex intermediate species. Triplex formation is rate limiting in 0.125 M KCl solution. In LiCl, each step of the association process depends on the strand concentration. Parallel reactions to formation of the fully matched canonical quadruplex may result in kinetically trapped mismatched quadruplexes making the canonical quadruplex practically inaccessible in particular at low temperature in KCl solution.     

Intramolecular quadruplex conformation of human telomeric DNA assessed with 125I-radioprobing

A repeated non-coding DNA sequence d(TTAGGG)_n is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter and intramolecular non-B-DNA conformations that may play an important role in biological processes. Two intramolecular structures of the telomeric oligonucleotide dAGGG(TTAGGG)₃, antiparallel and parallel, have been solved by NMR and X-ray crystallography. In both structures, the telomeric sequence adopts an intramolecular quadruplex structure that is stabilized by G-4 quartets, but the ways in which the sequence folds into the quadruplex are different. The folds of the human telomeric DNA were described as an anti-parallel basket-type and a parallel propeller-type. We applied ¹²⁵I-radioprobing to determine the conformation of the telomeric quadruplex in solution, in the presence of either Na⁺ or K⁺ ions. The probability of DNA breaks caused by decay of ¹²⁵I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks. The probability of breaks measured in the presence of Na⁺ and K⁺ were compared with the distances in basket-type and propeller-type quadruplexes obtained from the NMR and crystal structures. Our radioprobing data demonstrate that the antiparallel conformation was present in solution in the presence of both K⁺ and Na⁺. The preferable conformation in the Na⁺-containing solution was the basket-type antiparallel quadruplex whereas the presence of K⁺ favored the chair-type antiparallel quadruplex. Thus, we believe that the two antiparallel and the parallel conformations may coexist in solution, and that their relative proportion is determined by the type and concentration of ions.     

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K⁺ ions and only one in the presence of Na⁺ ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K⁺ but not Na⁺. Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na⁺ solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na⁺-containing environment. These structures, together with the coexistence of other conformations in Na⁺ solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K⁺ ions.     

A parallel stranded G‐quadruplex composed of threose nucleic acid (TNA)

G‐rich sequences can adopt four‐stranded helical structures, called G‐quadruplexes, that self‐assemble around monovalent cations like sodium (Na⁺) and potassium (K⁺). Whether similar structures can be formed from xeno‐nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α‐l ‐threofuranosyl nucleic acid) to adopt a four‐stranded helical structure based on a planar G‐quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution‐state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self‐assemble into stable G‐quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na⁺ and K⁺ ions, as G‐quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self‐assembling into stable G‐quadruplex structures.     

Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5′-AGGG(TTAGGG)₃] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na⁺ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K⁺ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K⁺ solutions. Sedimentation coefficients were determined experimentally in both Na⁺ and K⁺ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na⁺ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K⁺ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K⁺ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K⁺ solutions, but not in Na⁺ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K⁺ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K⁺ solution is not the one determined in the published crystalline state.     

Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

Interaction Energies and Dynamics of Alkali and Alkaline-Earth Cations in Quadruplex-DNA-Structures

We have investigated the nonbonded interaction energies and dynamical properties of different types of cations in quadruplex DNA structures using the GROMOS force field [1]. Quadruplex structures consist of planar guanine-quartets stacking together and causing the formation of a channel, large enought to enclose several cations (Figure 1). In recent years many experimental studies have indicated a prefered formation of this unusually stabel complexes with K⁺-ions. However, the high selectivity of this cation has not yet been understood [2].To determine the most stable coordination sites and the mobility of cations, we have calculated the pair potential energy of alkali and alkaline-earth cations along the helical axis of a model quadruplex structure (Figure 2). Our force field calculations indicate that small ions like Li⁺, Na⁺, Mg²⁺ and Ca²⁺ are free to move throught the channel. In contrast, for K⁺ and larger ions a high potential barrier appears, located in the plane of the tetramer unit. These findings are in agreement with data from X-ray crystallography, indicating that K⁺ cations are located between two planes while Na⁺ ions also can occupy coordination sites in the G-quartet plane.Considering solvent atoms in our calculations leads to the observation that a cation at the end of the quadruplex strongly interacts with one water molecule located near the entrance of the cage. Snapshots taken at different times of the MD simulation provide configurations which differ mainly in the position of this complexing water molecule. Moving away from the entrance of the cage causes a significant decrease of the potential barrier for K⁺ and smaller cage cations (Figure 3). For the larger ions the potential barrier is much higher than the thermal energy (not shown), preventing the cations from leaving and entering the cage.This conclusion is in agreement with results from our MD simulations. We followed the dynamics of different cations. While K⁺ is able to leave as well as to re-migrate into the channel (movie I), this was not observed for other types of cations. Figure 4 shows the time history of the positional fluctuations of potassium along the helical axis, and in Figure 5 we have monitored the distance between the O6-oxygen atoms of the outer G-quartet. It becomes clearly evident that the cation movement through the planes is correlated with the dynamic behaviour of the tetrameric planes. When the K⁺-ion penetrates the tetrameric unit to enter the quadruplex, the O6-O6-distance - a measure of size of the hole of the plane - increases. After a while the cation has reached the cage position and the G-quartet contracts to the initial value. That means the tetrameric planes perform a kind of breathing motion.For lithium ions we find a much higher mobility of the cation within the quadruplex channel. Two of three ions are leaving the cage instantaniously (not shown).Another indication of the experimentally observed much weaker complexation tendency of quadruplexes with lithium is the change in the distances of planes (a measure for the cage size) with time. While in the case of potassium the distance of planes is nearly the same for all three cages, for lithium the central occupied cage is much smaller than the cage in the starting structure, indicating that the DNA structure has to adjust its conformation to the cation size. On the other side the outer unoccupied cages are much greater and less stable. Due to this cation induced quadruplex deformation we observe an unwinding of the DNA-structure in the presens of lithium ions at longer simulation periods (400 ps).     

The Terminal Phosphate in d(pGpG) Reduces Self-Association

Abstract

An investigation of the self-association behavior of 2′-deoxy[5′-phosphate-guanylyl-(3′-5′)-guanosine] (d(pGpG)) in the presence of Na⁺ and K⁺ ions has been carried out by ¹H and ³¹PNMR and FTIR spectroscopy. A comparison has been made of the self-association behavior of d(pGpG) with that of the related dinucleotide d(GpG), which has been shown to form extended structures based on stacked G-tetrads. Chemically, d(pGpG) monomer differs from d(GpG) only by the addition of a phosphate at the 5′-OH of the sugar residue. It was found that the addition of the second phosphate interferes with self-association. A suitable counterion is all that is required by d(GpG) to induce the formation of large super structures, but for d(pGpG) a large excess of salt is needed to produce the same effect. However, once self-association occurs, d(pGpG) forms similar structures to d(GpG) and has nearly the same properties. For both compounds, the K⁺ ion induces a more stable structure than the Na⁺ ion. The ³¹P NMR chemical shift ranges of d(pGpG) were consistent with the reported data for a phosphodiester and terminal phosphate. The small change in the chemical shift of the terminal phosphate with increasing temperature suggests that no major change in the terminal phosphate conformation occurred upon self-association. It was concluded that the terminal phosphate did not result in steric hindrance to self-association, but that interference to self-association was due to electrostatic repulsion effects.     

Preferential binding of K+ ions in the selectivity filter at equilibrium explains high selectivity of K+ channels

K⁺ channels exhibit strong selectivity for K⁺ ions over Na⁺ ions based on electrophysiology experiments that measure ions competing for passage through the channel. During this conduction process, multiple ions interact within the region of the channel called the selectivity filter. Ion selectivity may arise from an equilibrium preference for K⁺ ions within the selectivity filter or from a kinetic mechanism whereby Na⁺ ions are precluded from entering the selectivity filter. Here, we measure the equilibrium affinity and selectivity of K⁺ and Na⁺ ions binding to two different K⁺ channels, KcsA and MthK, using isothermal titration calorimetry. Both channels exhibit a large preference for K⁺ over Na⁺ ions at equilibrium, in line with electrophysiology recordings of reversal potentials and Ba²⁺ block experiments used to measure the selectivity of the external-most ion-binding sites. These results suggest that the high selectivity observed during ion conduction can originate from a strong equilibrium preference for K⁺ ions in the selectivity filter, and that K⁺ selectivity is an intrinsic property of the filter. We hypothesize that the equilibrium preference for K⁺ ions originates in part through the optimal spacing between sites to accommodate multiple K⁺ ions within the selectivity filter.     

Competitive Binding of Mg2+ and Na+ Ions to Nucleic Acids: From Helices to Tertiary Structures

Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg²⁺/Na⁺ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg²⁺/Na⁺ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg²⁺/Na⁺ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg²⁺ over Na⁺ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg²⁺/Na⁺ concentrations. Furthermore, the local binding of Mg²⁺/Na⁺ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg²⁺/Na⁺ concentrations.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma     

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K⁺ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K⁺ solution is distinct from those reported on the 22 nt Tel22 in Na⁺ solution and in crystalline state in the presence of K⁺, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K⁺, regardless of the presence or absence of Na⁺. Furthermore, the addition of K⁺ readily converts the Na⁺-form conformation to the K⁺-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K⁺, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.     

KcsA 通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离子的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决.为了在分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能.计算结果表明,Rb+离子具有与K+离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K+离子的位垒高,因而所对应的通透率也就小于钾离子的通透率,而钠离子的的通透率仅仅是钾离子通透率的0.0067%.文中所涉及的系统仅仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统大小为41 000个原子.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.