Long-lived Water Molecules Research Articles

Introducing NMR strategies to define water molecules that drive metal binding in a transcriptional regulator

Staphylococcus aureus CzrA is a paradigmatic member of the ArsR family of transcriptional metalloregulators, which are critical for the bacterial response to stress. Zinc binding to CzrA, which induces DNA derepression, is entropically driven, as shown by calorimetry. A detailed equilibrium dynamics study of different allosteric states of CzrA revealed that zinc induces an entropy redistribution that controls for DNA binding regulation; however, this change in conformational entropy only accounts for a small net contribution to the total entropy. This difference between the change in conformational entropy vs. total entropy of zinc binding implies a significant contribution of solvent molecule rearrangements to this equilibrium. However, the absence of major structural changes suggests that solvent rearrangements occur mainly on the protein surface and/or from zinc desolvation, concomitant with a dynamical redistribution of conformational entropy. Previous results also suggest that zinc binding not only leads to a redistribution of protein internal dynamics, but also release of water molecules from the protein surface. In turn, these water molecules may make a significant contribution to the allosteric response that results in dissociation from the DNA.Quantifying the differential hydration of two conformational states that share very similar crystal structures and then correlating this with the protein's solvent entropy change constitutes an unresolved problem, even when thermodynamics suggest a significant contribution of solvent entropy. Here, we present different avenues to dissect hydration dynamics in a metal-binding transcriptional regulator that provide different insights into this complex problem. We explore primary solution NMR tools for probing protein–water interactions: the laboratory frame nuclear Overhauser effect (NOE) and its rotating frame counterpart (ROE) between long-lived water molecules and the protein residues. The wNOE/wROE ratio is a promising tool for the detection of hydration dynamics near the surface of a protein in a site-specific manner, minimizing contamination from bulk solvent. Molecular dynamics simulations and computational methods designed to provide a spatially resolved picture of solvent thermodynamics were also employed to provide a more complete panorama of solvent redistribution.

Correlation in Domain Fluctuations Navigates Target Search of a Viral Peptide along RNA.

Biological macromolecules often exhibit correlations in fluctuations involving distinct domains. This study decodes their functional implications in RNA-protein recognition and target-specific binding. The target search of a peptide along RNA in a viral TAR-Tat complex is closely monitored using atomistic simulations, steered molecular dynamics simulations, free energy calculations, and a machine-learning-based clustering technique. An anticorrelated domain fluctuation is identified between the tetraloop and the bulge region in the apo form of TAR RNA that sets a hierarchy in the domain-specific fluctuations at each binding event and that directs the succeeding binding footsteps. Thus, at each binding footstep, the dynamic partner selects an RNA location for binding where it senses a higher fluctuation, which is conventionally reduced upon binding. This event stimulates an alternate domain fluctuation, which then dictates sequential binding footstep/s and thus the search progresses. Our cross-correlation maps show that the fluctuations relay from one domain to another specific domain until the anticorrelation between those interdomain fluctuations sustains. Artificial attenuation of that hierarchical domain fluctuation inhibits specific RNA binding. The binding is completed with the arrival of a few long-lived water molecules that mediate slightly distant RNA-protein sites and finally stabilize the overall complex. The study underscores the functional importance of naturally designed fluctuating RNA motifs (bulge, tetraloop) and their interplay in dictating the directionality of the search in a highly dynamic environment.

Water-Triggered, Irreversible Conformational Change of SARS-CoV-2 Main Protease on Passing from the Solid State to Aqueous Solution.

The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1-ms-long molecular dynamics simulation performed by D. E. Shaw’s research group shows that it readily undergoes a symmetry-breaking event on passing from the solid state to aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analyzing this long simulation, we uncover a previously unrecognized role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling simulations performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.

Observation of Water Molecules within the Bimolecular d(G3CT4G3C)2 G-Quadruplex

G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.

High-Relaxivity Magnetic Resonance Imaging Contrast Agents Part 2

The observed relaxivity of gadolinium-based contrast agents has contributions from the water molecule(s) that bind directly to the gadolinium ion (inner-sphere water), long-lived water molecules and exchangeable protons that make up the second-sphere of coordination, and water molecules that diffuse near the contrast agent (outer-sphere). Inner- and second-sphere relaxivity can both be increased by optimization of the lifetimes of the water molecules and protons in these coordination spheres, the rotational motion of the complex, and the electronic relaxation of the gadolinium ion. We sought to identify new high-relaxivity contrast agents by systematically varying the donor atoms that bind directly to gadolinium to increase inner-sphere relaxivity and concurrently including substituents that influence the second-sphere relaxivity. Twenty gadolinium-1,4,7,10-tetraazacyclo-dodecane-N,N',N″,N'″-tetraacetato derivatives were prepared and their relaxivity determined in presence and absence of human serum albumin as a function of temperature and magnetic field. Data was analyzed to extract the underlying molecular parameters influencing relaxivity. Each compound had a common albumin-binding group and an inner-sphere donor set comprising the 4 tertiary amine N atoms from cyclen, an α-substituted acetate oxygen atom, 2 amide oxygen atoms, an inner-sphere water oxygen atom, and a variable donor group. Each amide nitrogen was substituted with different groups to promote hydrogen bonding with second-sphere water molecules. Relativities at 0.47 and 1.4 T, 37°C, in serum albumin ranged from 16.0 to 58.1 mM(-1)s(-1) and from 12.3 to 34.8 mM(-1)s(-1), respectively. The reduction of inner-sphere water exchange typical of amide donor groups could be offset by incorporating a phosphonate or phenolate oxygen atom donor in the first coordination sphere, resulting in higher relaxivity. Amide nitrogen substitution with pendant phosphonate or carboxylate groups increased relaxivity by as much as 88% compared with the N-methyl amide analog. Second-sphere relaxivity contributed as much as 24 and 14 mM(-1)s(-1) at 0.47 and 1.4 T, respectively. Water/proton exchange dynamics in the inner- and second-coordination sphere can be predictably tuned by choice of donor atoms and second-sphere substituents, resulting in high-relaxivity agents.

Relaxation of protons by radicals in rotationally immobilized proteins

Proton spin–lattice relaxation by paramagnetic centers may be dramatically enhanced if the paramagnetic center is rotationally immobilized in the magnetic field. The details of the relaxation mechanism are different from those appropriate to solutions of paramagnetic relaxation agents. We report here large enhancements in the proton spin–lattice relaxation rate constants associated with organic radicals when the radical system is rigidly connected with a rotationally immobilized macromolecular matrix such as a dry protein or a cross-linked protein gel. The paramagnetic contribution to the protein–proton population is direct and distributed internally among the protein protons by efficient spin diffusion. In the case of a cross-linked-protein gel, the paramagnetic effects are carried to the water spins indirectly by chemical exchange mechanisms involving water molecule exchange with rare long-lived water molecule binding sites on the immobilized protein and proton exchange. The dramatic increase in the efficiency of spin relaxation by organic radicals compared with metal systems at low magnetic field strengths results because the electron relaxation time of the radical is orders of magnitude larger than that for metal systems. This gain in relaxation efficiency provides completely new opportunities for the design of spin–lattice relaxation based contrast agents in magnetic imaging and also provides new ways to examine intramolecular protein dynamics.

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.

1H nuclear magnetic relaxation dispersion of Cu,Zn superoxide dismutase in the native and guanidinium-induced unfolded forms

Potentialities and limitations of the use of 1H NMRD technique for the characterization of the hydration properties of unfolded or partially folded states of proteins are discussed. The copper(I) form of monomeric Cu,Zn superoxide dismutase in its folded state and in the presence of 4 M guanidinium chloride is taken as case system. The dispersion profile, analyzed with an extended relaxation matrix analysis, indicates the presence of long-lived water molecules in the folded state. The observed increase in relaxation at high field upon addition of guanidinium chloride indicates an increase in the number of solvation protons interacting with the protein and exchanging with a time shorter than the protein reorientational time. The observed effect is consistent with an exposed protein surface of SOD in the presence of 4 M guanidinium chloride smaller than what could be expected for a random coil.

Stabilization of Internal Charges in a Protein: Water Penetration or Conformational Change?

The ionizable amino acid side chains of proteins are usually located at the surface. However, in some proteins an ionizable group is embedded in an apolar internal region. Such buried ionizable groups destabilize the protein and may trigger conformational changes in response to pH variations. Because of the prohibitive energetic cost of transferring a charged group from water to an apolar medium, other stabilizing factors must be invoked, such as ionization-induced water penetration or structural changes. To examine the role of water penetration, we have measured the 17O and 2H magnetic relaxation dispersions (MRD) for the V66E and V66K mutants of staphylococcal nuclease, where glutamic acid and lysine residues are buried in predominantly apolar environments. At neutral pH, where these residues are uncharged, we find no evidence of buried water molecules near the mutation site. This contrasts with a previous cryogenic crystal structure of the V66E mutant, but is consistent with the room-temperature crystal structure reported here. MRD measurements at different pH values show that ionization of Glu-66 or Lys-66 is not accompanied by penetration of long-lived water molecules. On the other hand, the MRD data are consistent with a local conformational change in response to ionization of the internal residues.

Trifluoroethanol-induced beta --> alpha transition in beta-lactoglobulin: hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion.

Alcohols, such as 2,2,2-trifluoroethanol (TFE), have been shown to induce a cooperative transition to an open helical structure in many proteins, but the underlying molecular mechanism has not been identified. Here, we employ the technique of magnetic relaxation dispersion (MRD) to study the TFE-induced beta --> alpha transition of beta-lactoglobulin at pH 2.4. Unlike traditional techniques that focus on protein secondary structure, the MRD method directly monitors the solvent, providing quantitative information about preferential solvation and solvent penetration and about the overall size and structural integrity of the protein. In this multinuclear MRD study, we use the (2)H and (17)O resonances to examine hydration and the (19)F resonance to study TFE. The transformation from the native to the helical state via an intermediate state at 300 K is found to be accompanied by a progressive expansion of the protein and loss of specific long-lived hydration sites. The observation of (17)O and (19)F dispersions from the helical state shows that water and TFE penetrate the protein. The MRD data indicate a strong accumulation of TFE at the surface as well as in the interior of the protein. At 277 K, BLG is much less affected by TFE, remaining in the native state at 16% TFE, but adopting a nonnative structure at 30% TFE. This nonnative structure is not penetrated by long-lived water molecules. The implications of these findings for the mechanism of TFE-induced structural transformations are discussed.

A high-resolution NMR study of long-lived water molecules in both oxidation states of a minimal cytochrome c.

The interaction of water with oxidized and reduced cytochrome c from the Gram-positive bacterium Bacillus pasteurii (a 71-amino acid long monoheme cytochrome) is investigated through CLEANEX experiments and (15)N-edited ePHOGSY and Tr-ROESY experiments. It appears that a water molecule gives rise to dipolar cross-relaxation with the amide protons of Gly74 and Ile75, with a residence time longer than 0.4 ns, to account for a negative NOE. Such water molecule is present in both the oxidized and reduced species and in the X-ray structure. It appears to have a structural role. Other possible roles are discussed by comparison with the water molecules present in other c-type cytochromes. The amide proton of Cys35 is found to exchange rapidly with the solvent in the oxidized but not in the reduced protein, at variance with H/D exchange experiments, which probe a different time scale. The present data confirm that electron-transfer proteins evolved to minimize reorganization energy upon change of the oxidation state, even though the consequent variation of charge of the metal ion may induce some changes in the structure and/or dynamics of the protein.

Protein Reorientation and Bound Water Molecules Measured by 1H Magnetic Spin-Lattice Relaxation

The water-proton spin-lattice relaxation rate constant, 1 /T 1, was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.

Structural parameterization of the binding enthalpy of small ligands.

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).

The solution structure of [d(CGC)r(amamam)d(TTTGCG)]2.

The solution structure and hydration of a DNA.RNA hybrid chimeric duplex [d(CGC)r(amamam)d(TTTGCG)]2 in which the RNA adenines were substituted by 2'-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2'-OMe-RNA.DNA junction adopted an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA.2'-OMe-RNA junction, adopted C1'-exo or C2'-endo conformations. The observed NOE intensity of 2'-O-methyl group to H1' proton of 4am at the DNA.2'-OMe-RNA junction is much weaker than those of 5am and 6am. The 2'-O-methyl group of 4am was found to orient towards the minor groove in the trans domain while the 2'-O-methyl groups of 5am and 6am were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1' protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]2, there were no long-lived water molecules found in [d(CGC)r(amamam)d(TTTGCG)]2. This is probably due to the hydrophobic enviroment created by the 2'-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2'-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5 degrees C to 51.9 degrees C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNA.RNA hybrid and chimeric duplexes with 2'-modified RNA residues.

Protein hydration and location of water molecules in oxidized horse heart cytochrome c by (1)H NMR.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by (1)H NMR spectroscopy. Two-dimensional, homonuclear ePHOGSY-NOESY experiments are used to map water-protein interactions. The detected NOEs reveal interactions between nonexchangeable protein protons and both water protons and labile protein protons which exchange with water protons. Among the many water molecules apparent in the X-ray structure, three have been identified with a residence time longer than 300 ps. One of them is located inside the distal heme cavity, in the deepest part of a hydration pathway extending toward the surface. The identification of hydrophilic regions and detection of three long-lived water molecules settles some ambiguities and provides a better representation of the water-protein interactions in oxidized cytochrome c.

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes.

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2), in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)](2)as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)](2). Long-lived water molecules with correlation time tau(c)longer than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations. The exchange rates for RNA C2'-OH were found to be approximately 5-20 s(-1). This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2)is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA. RNA hybrid and chimeric duplexes.

Hydration of [d(CGC)r(aaa)d(TTTGCG)] 2

We have studied the hydration and dynamics of RNA C2′-OH in a DNA·RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)] 2. Long-lived water molecules with correlation time τ c larger than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4′- endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1′- exo or C2′- endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2′-OH tends to orient toward the O3′ direction, forming a possible hydrogen bond with the 3′-phosphate group. The exchange rates for RNA C2′-OH were found to be around 5–20 s −1, compared to 26.7(±13.8) s −1 reported previously for the other DNA·RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)] 2, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1′ protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2′-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.

Characterization of monomeric and dimeric B domain of Staphylococcal protein A.

Both monomeric and dimeric constructs of the B domain of protein A from Staphylococcus aureus have been characterized by NMR, CD and fluorescence spectroscopy. The monomeric form of the protein was synthesized using a novel method incorporating the use of a recombinant, folded, chimeric protein. A comparison of the recombinant monomeric form with the commercially available dimeric form indicates that, although the dimer retains the integrity of the three-helix bundle structure present in the monomer, there are interdomain contacts in the dimeric form. A single long-lived water molecule in the hydrophobic core of the three-helix bundle of monomeric protein A may represent an important stabilizing factor for the three-helix bundle topology.

A molecular dynamics simulation of the flavin mononucleotide–RNA aptamer complex

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)–RNA aptamer. The simulated average structure maintains both cross-strand and intermolecular FMN–RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10–U12–A25 base triple and the A13–G24, A8–G28, and G9–G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long-lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long-lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long-lived “structural” water is at least two and in most cases three or four potential acceptor atoms. The 2′-OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2′H(n)⋮O4′(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2′-OH hydrogen atoms is approximately toward O3′ of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle γ in the trans orientation is found. A survey of all experimental RNA x-ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x-ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle γ in the trans orientation leads to a relatively short distance between 2′-OH(n) and O5′(n + 1), enabling hydrogen-bond formation. In this case the preferred orientation of the 2′-OH hydrogen atom is approximately toward O5′(n + 1). We find two relatively short and dynamically stable types of backbone–backbone next-neighbor contacts, namely C2′(H)(n)⋮O4′(n + 1) and C5′(H)(n + 1)⋮O2′(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 287–302, 1999

A molecular dynamics simulation of the flavin mononucleotide-RNA aptamer complex.

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)-RNA aptamer. The simulated average structure maintains both cross-strand and intermolecular FMN-RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10-U12-A25 base triple and the A13-G24, A8-G28, and G9-G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long-lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long-lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long-lived "structural" water is at least two and in most cases three or four potential acceptor atoms. The 2'-OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2'H(n) vertical ...O4'(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2'-OH hydrogen atoms is approximately toward O3' of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle gamma in the trans orientation is found. A survey of all experimental RNA x-ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x-ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle gamma in the trans orientation leads to a relatively short distance between 2'-OH(n) and O5'(n + 1), enabling hydrogen-bond formation. In this case the preferred orientation of the 2'-OH hydrogen atom is approximately toward O5'(n + 1). We find two relatively short and dynamically stable types of backbone-backbone next-neighbor contacts, namely C2'(H)(n) vertical ...O4'(n + 1) and C5'(H)(n + 1) vertical ...O2'(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures.