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 glycosyl 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.     

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

Abstract

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.     

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair

Osmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.     

Ion permeation through a narrow channel: using gramicidin to ascertain all-atom molecular dynamics potential of mean force methodology and biomolecular force fields

We investigate methods for extracting the potential of mean force (PMF) governing ion permeation from molecular dynamics simulations (MD) using gramicidin A as a prototypical narrow ion channel. It is possible to obtain well-converged meaningful PMFs using all-atom MD, which predict experimental observables within order-of-magnitude agreement with experimental results. This was possible by careful attention to issues of statistical convergence of the PMF, finite size effects, and lipid hydrocarbon chain polarizability. When comparing the modern all-atom force fields of CHARMM27 and AMBER94, we found that a fairly consistent picture emerges, and that both AMBER94 and CHARMM27 predict observables that are in semiquantitative agreement with both the experimental conductance and dissociation coefficient. Even small changes in the force field, however, result in significant changes in permeation energetics. Furthermore, the full two-dimensional free-energy surface describing permeation reveals the location and magnitude of the central barrier and the location of two binding sites for K(+) ion permeation near the channel entrance--i.e., an inner site on-axis and an outer site off-axis. We conclude that the MD-PMF approach is a powerful tool for understanding and predicting the function of narrow ion channels in a manner that is consistent with the atomic and thermally fluctuating nature of proteins.     

Extra stable 2',5'-linked RNA loops

We prepared hairpins that differ in the connectivity of phosphodiester linkages in the loop (RNA vs 2', 5'-RNA). We find that the stability of the extra stable RNA hairpin 5'-rGGAC(UUCG)GUCC-3' is the same as that observed for the hairpin containing a 2',5'RNA loop, i.e. 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U2'p5'U2'p5' C2'p5'G2'p5'). Also significant is the finding that when the stem is duplex DNA, duplex 2',5'-RNA, or DNA:2',5'-RNA, hairpins with the UUCG loop are more stable than those with UUCG loop.     

The nature of preferred hairpin structures in 16S-like rRNA variable regions.

Variable length hairpins in 16S-like rRNA show a predominance for tetra-loops, its degree correlates with the protein content of the ribosome. The number of base-pairs adjacent to the loop (the tip size) and the nearest neighbor composition contribute to the stability of hairpin structures. The average tip size in length variable hairpins correlates with the thermophilicity of the organism, i.e. in temperate environments less stable stem structures are tolerated or even necessary. The most abundant loop families UUCG, GCAA, and CUUG occur most frequently at loop sizes 3, 2, and 7, respectively. Short tips of size less than or equal to 4 generally prefer nearest-neighbor combinations that result in CCC-GGG. Loop-specific tipmost nearest neighbors are revealed at longer tips: CUC(UUCG)GAG, GUA(GCAA)UAC with a maximum at tip sizes 5-6, and GWG(CUUG)CWC. Conserved hairpins, however, prefer variants of the UUCG and GCAA motifs with additional purines. Minor loop families and single motifs such as UUUA, UUUU, CUUGU, UUCGG, and UUU are investigated for preferable tip sizes and nearest-neighbor composition. Specific features are revealed for prominent hexa-loops.     

Folding simulations of three proteins having all α-helix,all β-strand and α/β-structures

We performed folding simulations of three proteins using four force fields, AMBER parm96, AMBER parm99, CHARMM 27 and OPLS-AA/L, in order to examine the features of these force fields. We studied three proteins, protein A (all α-helix), cold-shock protein (all β-strand) and protein G (α/β-structures), for the folding simulations. For the simulation, we used the simulated annealing molecular dynamics method, which was performed 50 times for each protein using the four force fields. The results showed that the secondary-structure-forming tendencies are largely different among the four force fields. AMBER parm96 favours β-bridge structures and extended β-strand structures, and AMBER parm99 favours α-helix structures and 3₁₀-helix structures. CHARMM 27 slightly favours α-helix structures, and there are also π-helix and β-bridge structures. OPLS-AA/L favours α-helix structures and 3₁₀-helix structures.     

Dielectric screening effect of electronic polarization and intramolecular hydrogen bonding

Recent site‐resolved hydrogen exchange measurements have uncovered significant discrepancies between simulations and experimental data during protein folding, including the excessive intramolecular hydrogen bonds in simulations. This finding indicates a possibility that intramolecular charge–charge interactions have not included sufficient dielectric screening effect of the electronic polarization. Scaling down peptide atomic charges according to the optical dielectric constant is tested in this study. As a result, the number of intramolecular hydrogen bonds is lower than using unscaled atomic charges while reaching the same levels of helical contents or β‐hairpin backbone hydrogen bonds, because van der Waals interactions contribute substantially to peptide folding in water. Reducing intramolecular charge–charge interactions and hydrogen bonding increases conformational search efficiency. In particular, it reduces the equilibrium helical content in simulations using AMBER force field and the energy barrier in folding simulations using CHARMM force field.     

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.     

Understanding the thermodynamic stability of an RNA hairpin and its mutant.

The thermodynamic stability of RNA hairpin loops has been a subject of considerable interest in the recent past (Wimberly et al., 1991). There have been experimental reports indicating that the hairpins with a C(UUCG)G loop sequence are thermodynamically very stable (Wimberly et al., 1991). We used the solution structure of GGAC(UUCG)GUCC (Cheong et al., 1990; Varani et al., 1991) as the starting conformation in our attempt to understand its thermodynamic stability. We carried out molecular dynamics/free energy simulations to understand the basis for the destabilization of the C(UUCG)G loop by mutating cytosine (C7)-->uracil. Because of the limited length of simulation and the presence of kinetic barriers (solvent intervention) to the uracil-->cytosine mutation, all of our computed free energy differences are based on multiple forward simulations. Based on these calculations we find that the cytosine-->uracil mutation in the loop destabilizes it by approximately 1.5kcal/mol relative to that of the reference state, an A-form RNA but with cytosine (C7) looped out. This is the same sign and magnitude as that observed in the thermodynamic studies carried out by Varani et al.(1991). We have carried out free energy component analysis to understand the effect of mutating the cytosine residue to uracil on the thermodynamic stability of the C(UUCG)G hairpin loops. Our calculations show that the most significant contribution to the stability is from the phosphate group linking U5 and U6, which favors the cytosine residue over uracil by about 6.0 kcal/mol. The residues U5, U6, and G8 in the loop region also contribute significantly to the stability. The contributions from the salt and solvent compensate each other, indicating the dynamic nature of interactions of the environment with the nucleic acid system and the coupling between these two components.     

Effects of base substitutions in an RNA hairpin from molecular dynamics and free energy simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.     

Preference for ribose over deoxyribose in loop-closing base pairs of extra stable nucleic acid hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5'-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, "extra stable" RNA hairpin structure. Recently, we showed that hairpins containing a 2',5'-linked (UUCG) loop instead of the native 3',5'-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368-12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC4 and rG9) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3',5'-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2',5'-UUCC loop and, in fact, they may be replaced altogether (ribose--> deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

稳定的RNA发夹结构及其生物学功能

以UNCG、GNRA、CUUG(N=A、U、C或G,R=G或A)为端环能够形成稳定的、保守的发夹结构。高分辨率的溶液结构、晶体结构和计算机模拟等方法从原子水平上解析了这些发夹特殊的结构特征。在体内,它们发挥着重要的生物学功能:在折叠过程中作为折叠的起始位置帮助组织RNA分子正确折叠;与核酸受体结合参与三级相互作用;与蛋白质发生相互作用;阻止逆转录酶的延伸等等。另外,由于C(UUCG)G发夹极其稳定的特征,在体外RNA分子的实验测定中它还是稳定核酸结构的理想工具。这些稳定的发夹广泛分布于体内rRNA、催化RNA和非编码mRNA中。但在对人类编码区mRNA结构特征的研究当中,却未发现C(UUCG)G发夹。     

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability.

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.     

Sequence-dependent conformational differences of small RNAs revealed by native gel electrophoresis.

In this study, we use native polyacrylamide gel electrophoresis and one-dimensional NMR spectroscopy to analyze small RNA hairpins containing a UUCG tetraloop. The aggregation state of one RNA 16-mer (5'-CGGCUUCGGUCGACCA-3') in the presence of Mg(2+) was confirmed by laser light scattering. Although it is widely known in the RNA field that some RNAs tend to aggregate, especially when present at high concentrations, the sequence elements responsible for this effect are rarely identified. In this work, we show that Mg(2+)-induced aggregation of the 16-mer RNA hairpin is sensitive to the presence of the 3'-terminal base and a specific 2'-hydroxyl group. Our study highlights the fact that even small changes in a particular RNA sequence can increase its tendency to undergo Mg(2+)-dependent aggregation in an unpredictable manner. Our analysis also shows that native gel electrophoresis is a sensitive probe of RNA conformation with the capability to detect differences apparently caused by subtle base stacking effects at the ends of helices.     

40 Probing single molecule RNA folding using temperature and force

Nucleic acids can be unfolded either by temperature, such as in UV melting, or by mechanical force using optical tweezers. In UV melting experiments, the folding free energy of nucleic acids at mesophilic temperatures are extrapolated from unfolding occurring at elevated temperatures. Additionally, single molecule unfolding experiments are typically performed only at room temperature, preventing calculation of changes in enthalpy and entropy. Here, we present temperature-controlled optical tweezers suitable for studying folding of single RNA molecules at physiological temperatures. Constant temperatures between 22 and 37?°C are maintained with an accuracy of 0.1?°C, whereas the optical tweezers display a spatial resolution of ～1?nm over the temperature range. Using this instrument, we measured the folding thermodynamics and kinetics of a 20-base-pair RNA hairpin by force-ramp and constant force experiments. Between 22 and 37?°C, the hairpin unfolds and refolds in a single step. Increasing temperature decreases the stability of the hairpin and thus decreases the force required to unfold it. The equilibrium force, at which unfolding and refolding rates are equal, drops ～1?pN as temperature increases every 5?°C. At each temperature, the folding energy can be quantified by reversible work done to unfold the RNA and from the equilibrium constant at constant forces. Over the experimental temperature range, the folding free energy of the hairpin depends linearly on temperature, indicating that ΔH is constant. The measured folding thermodynamics are further compared with the nearest neighbor calculations using Turner’s parameters of nucleic acid folding energetics.     

Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

-1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base-base and base-sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in -1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ～30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.     

Incorporating global features of RNA motifs in predictions for an ensemble of secondary structures for encapsidated MS2 bacteriophage RNA

The secondary structure of encapsidated MS2 genomic RNA poses an interesting RNA folding challenge. Cryoelectron microscopy has demonstrated that encapsidated MS2 RNA is well-ordered. Models of MS2 assembly suggest that the RNA hairpin-protein interactions and the appropriate placement of hairpins in the MS2 RNA secondary structure can guide the formation of the correct icosahedral particle. The RNA hairpin motif that is recognized by the MS2 capsid protein dimers, however, is energetically unfavorable, and thus free energy predictions are biased against this motif. Computer programs called Crumple, Sliding Windows, and Assembly provide useful tools for prediction of viral RNA secondary structures when the traditional assumptions of RNA structure prediction by free energy minimization may not apply. These methods allow incorporation of global features of the RNA fold and motifs that are difficult to include directly in minimum free energy predictions. For example, with MS2 RNA the experimental data from SELEX experiments, crystallography, and theoretical calculations of the path for the series of hairpins can be incorporated in the RNA structure prediction, and thus the influence of free energy considerations can be modulated. This approach thoroughly explores conformational space and generates an ensemble of secondary structures. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.