Tertiary Structure Formation Research Articles

Physicochemical Biology: Conquered Boundaries and New Horizons

In this paper, we shall consider the main evolutionary stages that occurred within the field of physicochemical biology during the 20th century, following the determination of the tertiary structure of DNA by Watson and Crick and the subsequent successes in the X-ray structural analysis of biopolymers. The authors' ideas on the pre-emptive problems and the methods used in physicochemical biology in the 21st century are also presented, including an investigation of the dynamics of biochemical processes, studies of the functions of unstructured proteins, as well as single-molecule investigations of enzymatic processes and of biopolymer tertiary structure formation.

B lineage-specific deletion of Bim leads to lymphoproliferative and autoimmune disease (159.23)

Abstract Autoreactive B cells are known to contribute to the onset and progression of a variety of autoimmune diseases including systemic lupus erythematosus (SLE). To prevent this, the B cell receptor (BCR) exerts specificity-based quality control and B cell activating factor receptor (BAFF-R) maintains homeostasis primarily through controlling apoptosis. An appropriate balance in the levels of the pro- and anti-apoptotic members of the Bcl-2 family is central to this process. Using mice with B lineage-specific deletion of the pro-apoptotic gene Bim (B-bim-/-), we showed that B cell autonomous dysregulation of apoptosis is sufficient to cause systemic autoimmune disease with lymphoproliferation. B-bim-/- mice display progressive splenomegally and lymphoadenopathy with a significant increase in B cells as well as an overall increase in innate and T cells which display markers of activation. B-bim-/- mice had higher serum levels of autoantibodies and proinflammatory cytokines. These mice also displayed increased lymphocyte proliferation and infiltration of the salivary gland, lung and liver. Furthermore, accumulation of lymphocytes in the B-bim-/- mice results in the formation of tertiary lymphoid structures. These results suggest that apoptosis resistant B-bim-/- B cells can acquire an activated phenotype and become pathogenic, culminating in the activation of T cells and the generation of a pro-inflammatory microenvironment leading to systemic autoimmunity. In addition, massive lymphoproliferation and inflammation may predispose B-bim-/- mice to B cell and other hematopoietic malignancies.

Low Folding Cooperativity of Hp35 Revealed by Single-Molecule Force Spectroscopy and Molecular Dynamics Simulation

Some small proteins, such as HP35, fold at submicrosecond timescale with low folding cooperativity. Although these proteins have been extensively investigated, still relatively little is known about their folding mechanism. Here, using single-molecule force spectroscopy and steered molecule dynamics simulation, we study the unfolding of HP35 under external force. Our results show that HP35 unfolds at extremely low forces without a well-defined unfolding transition state. Subsequently, we probe the structure of unfolded HP35 using the persistence length obtained in the force spectroscopy. We found that the persistence length of unfolded HP35 is around 0.72 nm, >40% longer than typical unstructured proteins, suggesting that there are a significant amount of residual secondary structures in the unfolded HP35. Molecular dynamics simulation further confirmed this finding and revealed that many native contacts are preserved in HP35, even its two ends have been extended up to 8 nm. Our results therefore suggest that retaining a significant amount of secondary structures in the unfolded state of HP35 may be an efficient way to reduce the entropic cost for the formation of tertiary structure and increase the folding speed, although the folding cooperativity is compromised. Moreover, we anticipate that the methods we used in this work can be extended to the study of other proteins with complex folding behaviors and even intrinsically disordered ones.

Analysis and Antioxidant Capacity of Anthocyanin Pigments. Part II: Chemical Structure, Color, and Intake of Anthocyanins

Anthocyanins belong to a large group of secondary plant metabolites collectively known as flavonoids, a subclass of the polyphenol family. They are a group of very efficient bioactive compounds that are widely distributed in plant food. Anthocyanins occur in all plant tissues, including leaves, stems, roots, flowers, and fruits. Research on phenolic compounds through the last century, from the chemical, biochemical, and biological points of view, has focused mainly on the anthocyanins. Anthocyanins have structures consisting of two aromatic rings linked by three carbons in an oxygenated heterocycle (i.e., a chromane ring bearing a second aromatic ring in position 2). The basic chromophore of anthocyanins is the 7-hydroxyflavilyum ion. Anthocyanin pigments consist of two or three chemical units: an aglycon base or flavylium ring (anthocyanidin), sugars, and possibly acylating groups. Only six of the different anthocyanidins found in nature occur frequently and are of dietary importance: cyanidin, delphinidin, petunidin, peonidin, pelargonidin, and malvidin. Each aglycon may be glycosilated or acylated by different sugars and aromatic or aliphatic acids, yielding over 600 different anthocyanins reported from plants. The sugar moiety is typically attached at the 3-position on the C-ring or the 5-position on the A-ring. The chromophore of eight conjugate double bonds carrying a positive charge on the heterocyclic oxygen ring is responsible for the intensive red-orange to blue-violet color produced by anthocyanins under acidic conditions. Anthocyanins occur in solution as a mixture of different secondary structures: flavylium ion, a quinoidal base, a carbinol base, and a chalcone pseudobase. Self-association, intermolecular, and intramolecular co-pigmentation of anthocyanins leads to the formation of tertiary structures through varying stabilization mechanisms. Anthocyanin composition has been used as a botanical tool for taxonomic classification of plants. In addition, anthocyanin profiles of fruits and vegetables allow detecting adulteration of anthocyanin-based products and are indicators of product quality. Anthocyanins are common components of the human diet, as they are present in many foods, fruits, and vegetables, especially in berries. Moreover, anthocyanins have an antioxidant activity, depending to a large extent upon their chemical structure. Many epidemiological studies have shown the benefits of a diet rich in fruit and vegetables to human health, and for the prevention of various diseases associated with oxidative stress, such as cancer and cardiovascular diseases. Anthocyanin-rich extracts are increasingly attractive to the food industry as natural alternatives to synthetic FD&C dyes and lakes, because of their coloring properties. Anthocyanins are also one of the nine European Union-designated natural color classes. Various adverse effects on health have frequently been attributed to synthetic antioxidants. For these reasons, currently, there is a trend towards relying on antioxidants derived from natural products. Anthocyanins act as antioxidants both in the foodstuffs in which they are found and in the organism after intake of these foods. This review, like the first one of the series, intends to reflect the interdisciplinary nature of the research that is currently carried out in this prolific area.

Folding Models of Mini-Protein FSD-1

We have carried out all-atom action-derived molecular dynamics (ADMD) folding simulations of the full-size FSD-1. FSD-1 is a designed mini-protein of 28 residues containing both α and β secondary structure elements. Multiple folding pathways are found for FSD-1, which is consistent with existing computational studies. Hydrophobic collapse is observed first, and then subsequent folding events proceeds by forming either α-helix or β-hairpin. Concurrent formation of the full tertiary structure and the secondary structure elements of α-helix and β-hairpin is observed. The folding pathway of FSD-1 elucidated by ADMD simulations does not follow the scenario of the framework model. ADMD simulations provide significant insights for the general mechanisms of protein folding and conformational changes.

ChemInform Abstract: The Interplay of Turn Formation and Hydrophobic Interactions on the Early Kinetic Events in Protein Folding

AbstractReview: 67 refs.

Dimerization of Amino Acid Side Chains: Lessons from the Comparison of Different Force Fields.

The interactions between amino acid side chains govern protein secondary, tertiary, and quaternary structure formation. For molecular modeling approaches to be able to realistically describe these phenomena, the underlying force fields have to represent these interactions as accurately as possible. Here, we compare the side chain-side chain interactions for a number of commonly used force fields, namely the all-atom OPLS, the united-atom GROMOS, and the coarse-grain MARTINI force field. We do so by calculating the dimerization free energies between selected pairs of side chains and structural characterization of their binding modes. To mimic both polar and nonpolar environments, the simulations are performed in water, n-octanol, and decane. In general, reasonable correlations are found between all three force fields, with deviations on the order of 1 kT in aqueous solvent. In apolar solvent, however, significantly larger differences are found, especially for charged amino acid pairs between the OPLS and GROMOS force fields, and for polar interactions in the MARTINI force field in comparison to the higher resolution models. Interestingly, even in cases where the dimerization free energies are similar, the binding mode may differ substantially between the force fields. This was found to be especially the case for aromatic residues. In addition to the inter-force-field comparison, we compared the various force fields to a knowledge-based potential. The two independent approaches show good correlation in aqueous solvent with an exception of aromatic residues for which the interaction strength is lower in the knowledge-based potentials.

The impact of isomers of hemiaminal-1,2,4-triazole conjugates differently substituted in the phenyl ring and their Cu2+ complexes on the catalytic activity of the antigenomic delta ribozyme

The ability of four stable hemiaminals differently substituted in the phenyl ring and their complexes with Cu2+ ions to inhibit catalytic cleavage of the antigenomic delta ribozyme was compared. The hemiaminals were novel chiral derivatives of 1,2,4-triazole [i.e. (2,4-dinitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2,4-dnbald), (2-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2-nbald), (3-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (3-nbald) and (4-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (4-nbald)]. The complexes of nbalds with Cu2+ were characterized using UV and EPR methods and additionally, the formation of 2,4-dnbald–Cu2+ complex with CuL2 stoichiometry was confirmed by mass spectrometry.The data suggest that there are two ways in which nbalds and their Cu2+ complexes can influence catalytic cleavage of antigenomic delta ribozyme. The coordinated Cu2+ ions may play the role of new cationic ligands increasing the affinity of the complexes to the ribozyme. Such situation occurs in the case of 2- and 2,4-nbald. Their Cu2+ complexes decrease ribozyme cleavage rates twice more efficiently than uncomplexed compounds. Moreover, the Cu2+ complexes displace the catalytic divalent metal ions from their strong binding sites located in the ribozyme J4/2 region as shown by the Pb2+-induced cleavage approach. On the other hand, 3- and 4-nbald inhibit catalysis more strongly as compared to 2-nbald and 2,4-dnbald but the ribozyme cleavage rates are changed only slightly upon Cu2+ complexation. The mechanism of ribozyme inhibition by interfering with the formation of a correct ribozyme tertiary structure seems to operate in this case.

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family.

Acceleration of Disulfide-Coupled Protein Folding by Positively Charged Glutathione Derivatives

Protein folding is a spontaneous process under appropriate condition and results in the formation of the native tertiary structure. However, disulfide-containing proteins require a redox system to form the correct disulfide pairings. Glutathione plays an important role in the formation of disulfide bonds in proteins in the endoplasmic reticulum. Because of this, it is also widely used as a redox reagent in the disulfide-coupled folding of proteins. However, the mechanism of the disulfide-coupled folding of proteins by glutathione is not completely understood. Therefore, to examine the glutathione mediated-folding of proteins containing disulfide bonds, a series of glutathione derivatives, Glu-Cys-Arg (ECR) and Arg-Cys-Gly (RCG) were prepared and their participation in protein folding was examined using lysozyme and prouroguanylin as model proteins.The folding recovery of lysozyme using the oxidized and reduced forms of RCG was higher than that of the typically used glutathione redox system, while the results for ECR were similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin indicated that the folding velocity using the RCG system was approximately 3 fold faster than that of the glutathione system. Furthermore, folding experiments using only the oxidized form of RCG revealed that prouroguanylin was converted into the native conformation more efficiently by RCG, compared to glutathione. We therefore conclude that the RCG system is superior to the glutathione system for protein folding because of its ability to accelerate the disulfide exchange reaction and its ability to mediate the formation of native disulfide bonds in proteins. In this paper, we report on the use of the positively charged glutathione molecule in disulfide-coupled protein folding, which results in an improved folding recovery and permits the formation of the correct tertiary structure of the target proteins.

Modulating Transmembrane α-Helix Interactions through pH

The folding and assembly of α-helical integral membrane proteins into functional molecules is largely mediated by the lateral interactions of its transmembrane (TM) α-helices where sequence motifs often maximize van der Waals packing and/or polar interactions at sites of helix-helix contact. These interactions play a major role in determining the correct formation of tertiary and quaternary structure for these hydrophobic proteins. We have previously shown that a Lys-tagged TM2 α-helix of myelin proteolipid protein (PLP), of parent sequence, KKKK-AFQYVIYGTASFFFLYGALLLAEGF-KKKK, is capable of self-dimerization in the membrane mimetic detergent sodium dodecylsulfate (SDS). In the present work, we use protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer to demonstrate that the oligomeric state of this peptide can be modulated by pH. Specifically, as the pH is increased, the peptide shifts from dimer to monomer. Mutational analysis reveals that this functionality is due to the C-terminal glutamic acid. Our overall results emphasize the potential influence of surrounding residues on the interaction strength of TM helix-helix contact sites that mediate oligomerization and may have implications for the control of biologically relevant events involving helix-helix interactions in some membrane proteins.

The interplay of turn formation and hydrophobic interactions on the early kinetic events in protein folding

While both turn formation and hydrophobic interactions play dominant roles in the initiation of protein folding, their individual contributions to the folding kinetics and to the structural stability of the protein still remain poorly understood. Here, we applied a photolabile linker to "cage" some important structural motifs, including both α-helices and β-sheets, into their non-native states. These "caged" structural motifs are then relaxed by laser-flash photolysis and their refolding events followed by photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD). These experiments, combined with our previous results, revealed that spontaneous α-helix formation can occur extremely rapidly (10(8)-10(9) s(-1)) if the process is driven solely by turn formation followed by helix propagation. However, if sequestering of the side chains of hydrophobic amino acid residues participates in the refolding process, which may provide additional driving force beyond that afforded by turn formation alone, the refolding rate will be retarded, often by many orders of magnitude. This is usually the case in the formation of three-stranded β-sheets (10(7)-10(8) s(-1)) and β-hairpins (10(5)-10(6) s(-1)). Thus, we propose that proteins take advantage of the hierarchy of timescales associated with either turn formation, hydrophobic interactions, or global collapse of tertiary structure to accomplish the folding process in an orderly fashion, as these events are sufficiently separated in time and do not interfere with one another.

FRET Investigation of Membrane Protein Folding: Evolution of Tertiary Structure of Soluble and Transmembrane Domains during Folding into Synthetic Bilayers

This paper describes an investigation of the folding mechanisms of an integral membrane protein. A key goal is to determine the role of the soluble domain during the insertion of a transmembrane domain into synthetic bilayers. Towards this end, we report Forster resonance energy transfer (FRET) efficiencies between donor (tryptophan) and acceptor (1,5-IAEDANS) pairs that are located on the transmembrane and soluble domains of outer membrane protein A (OmpA). The FRET efficiencies are correlated to the evolution of distances and tertiary structure under the assumption of orientational averaging. Analysis of the kinetics reveals that the full-length protein, which contains both soluble and transmembrane domains, displays slower folding rates compared to the truncated variant, which is comprised of the transmembrane domain only. This difference in rates may reflect an increase in the number of kinetic traps during folding, or indicate alternate pathways. These measurements of the formation of tertiary structure and the role of a soluble domain on the kinetics of folding may aid in the elucidation of the mechanisms of membrane protein folding and dynamics.

Site-specific platinum(II) cross-linking in a ribozyme active site.

The function of RNA depends on its ability to adopt complex and dynamic structures, and the incorporation of site-specific cross-linking probes is a powerful method for providing distance constraints that are valuable in RNA structural biology. Here we describe a new RNA-RNA cross-linking strategy based on Pt(II) targeting of specific phosphorothioate substitutions. In this strategy cis-diammine Pt(II) complexes are kinetically recruited and anchored to a phosphorothioate substitution embedded within a structured RNA. Substitution of the remaining exchangeable Pt(II) ligand with a nucleophile supplied by a nearby RNA nucleobase results in metal-mediated cross-links that are stable during isolation. This type of cross-linking strategy was explored within the catalytic core of the Hammerhead ribozyme (HHRz). When a phosphorothioate substitution is installed at the scissile bond normally cleaved by the HHRz, Pt(II) cross-linking takes place to nucleotides G8 and G10 in the ribozyme active site. Both of these positions are predicted to be within ~8 Å of a phosphorothioate-bound Pt(II) metal center. Cross-linking depends on Mg(2+) ion concentration, reaching yields as high as 30%, with rates that indicate cation competition within the RNA three-helix junction. Cross-linking efficiency depends on accurate formation of the HHRz tertiary structure, and cross-links are not observed for RNA helices. Combined, these results show promise for using kinetically inert Pt(II) complexes as new site-specific cross-linking tools for exploring RNA structure and dynamics.

Abstract C141: Screening of novel RNA aptamer(s) that selectively bind(s) to prostatic acid phosphatase.

Abstract Prostate cancer is becoming one of the most popular non-skin cancers in Korea due to continued increase of western life styles. Since earlier detection of prostate cancer can lead to complete cure, a lot of studies have been done on prostate cancer markers such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP) and prostate stem cell antigen (PSCA). Among these biomarkers, PAP is gaining renewed interest because of its potential merits in diagnosis and therapeutics. PAP, which was initially identified in 1935, is an abundant enzyme that is synthesized in prostate epithelial cells and has enzymatic activity in acidic conditions. PAP expression is androgen-independent and increases in proportion to prostate cancer progression. Indeed, PAP expression level has high correlation with cause-specific survival and its dephosphorylation activity is believed to play an important role in the incidence and development of prostate cancer. Here, we screened the in vitro transcribed RNA libraries to get aptamer(s) that can selectively bind to PAP. Aptamers are oligonucleic acid molecules that bind to a specific target through formation of tertiary structures. These chemical antibodies can bind their target molecules with a high affinity and specificity and have the advantage of being less immunogenic in vivo. For in vitro selection, the ectracellular domain of PAP were cloned from PC3 cells and subjected to 6 rounds of SELEX procedure. This resulted in enrichment of the RNA aptamers that bound to the PAP ectodomain with 3-fold higher affinity than the library control as evidenced by real-time RT-PCR and gel shift assay. Sequence analysis of the aptamers revealed three distinct groups (G1, G2, N) having variable sequences within the region comprising nucleotides 43 to 87. The Kd values of the best clones with the highest affinity were within 120nM. These clones could also bind to the PAP-positive cells such as PC3 and LNCaP cells. Interestingly, 2'-OH-modification of the aptamers led to loss of binding, implying that 2'-F modification may be essential in binding to PAP. Characterization of these aptamers is currently underway and initial minimization results suggest that the structure coming from the 50 nucleotides is essential for binding to PAP. With further optimization, these PAP RNA aptamers could find wide application in theragonosis of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C141.

Characterization of Chemokines and Adhesion Molecules Associated with T cell Presence in Tertiary Lymphoid Structures in Human Lung Cancer

De novo formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies.

The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks

RNA molecules adopt specific three-dimensional structures critical to their function. Many essential metabolic processes, including protein synthesis and RNA splicing, are carried out by RNA molecules with elaborate tertiary structures (e.g. 3QIQ, right). Indeed, the ribosome and self-splicing introns are complex RNA machines. But even the coding regions in messenger RNAs and viral RNAs are flanked by highly structured untranslated regions, which provide regulatory information necessary for gene expression. RNA tertiary structure is defined as the three-dimensional arrangement of RNA building blocks, which include helical duplexes, triple-stranded structures, and other components that are held together through connections collectively termed RNA tertiary interactions. The structural diversity of these interactions is now a subject of intense investigation, involving the techniques of NMR, X-ray crystallography, chemical genetics, and phylogenetic analysis. At the same time, many investigators are using biophysical techniques to elucidate the driving forces for tertiary structure formation and the mechanisms for its stabilization. RNA tertiary folding is promoted by maximization of base stacking, much like the hydrophobic effect that drives protein folding. RNA folding also requires electrostatic stabilization, both through charge screening and site binding of metals, and it is enhanced by desolvation of the phosphate backbone. In this Account, we provide an overview of the features that specify and stabilize RNA tertiary structure. A major determinant for overall tertiary RNA architecture is local conformation in secondary-structure junctions, which are regions from which two or more duplexes project. At junctions and other structures, such as pseudoknots and kissing loops, adjacent helices stack on one another, and these coaxial stacks play a major role in dictating the overall architectural form of an RNA molecule. In addition to RNA junction topology, a second determinant for RNA tertiary structure is the formation of sequence-specific interactions. Networks of triple helices, tetraloop-receptor interactions, and other sequence-specific contacts establish the framework for the overall tertiary fold. The third determinant of tertiary structure is the formation of stabilizing stacking and backbone interactions, and many are not sequence specific. For example, ribose zippers allow 2'-hydroxyl groups on different RNA strands to form networks of interdigitated hydrogen bonds, serving to seal strands together and thereby stabilize adjacent substructures. These motifs often require monovalent and divalent cations, which can interact diffusely or through chelation to specific RNA functional groups. As we learn more about the components of RNA tertiary structure, we will be able to predict the structures of RNA molecules from their sequences, thereby obtaining key information about biological function. Understanding and predicting RNA structure is particularly important given the recent discovery that although most of our genome is transcribed into RNA molecules, few of them have a known function. The prevalence of RNA viruses and pathogens with RNA genomes makes RNA drug discovery an active area of research. Finally, knowledge of RNA structure will facilitate the engineering of supramolecular RNA structures, which can be used as nanomechanical components for new materials. But all of this promise depends on a better understanding of the RNA parts list, and how the pieces fit together.

Evidence for a Thermodynamically Distinct Mg2+ Ion Associated with Formation of an RNA Tertiary Structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ∼-3 kcal/mol of the total Mg(2+) interaction free energy of ∼-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.

RNA Folding Pathways and the Self-Assembly of Ribosomes

Many RNAs do not directly code proteins but are nonetheless indispensable to cellular function. These strands fold into intricate three-dimensional shapes that are essential structures in protein synthesis, splicing, and many other processes of gene regulation and expression. A variety of biophysical and biochemical methods are now showing, in real time, how ribosomal subunits and other ribonucleoprotein complexes assemble from their molecular components. Footprinting methods are particularly useful for studying the folding of long RNAs: they provide quantitative information about the conformational state of each residue and require little material. Data from footprinting complement the global information available from small-angle X-ray scattering or cryo-electron microscopy, as well as the dynamic information derived from single-molecule Förster resonance energy transfer (FRET) and NMR methods. In this Account, I discuss how we have used hydroxyl radical footprinting and other experimental methods to study pathways of RNA folding and 30S ribosome assembly. Hydroxyl radical footprinting probes the solvent accessibility of the RNA backbone at each residue in as little as 10 ms, providing detailed views of RNA folding pathways in real time. In conjunction with other methods such as solution scattering and single-molecule FRET, time-resolved footprinting of ribozymes showed that stable domains of RNA tertiary structure fold in less than 1 s. However, the free energy landscapes for RNA folding are rugged, and individual molecules kinetically partition into folding pathways that lead through metastable intermediates, stalling the folding or assembly process. Time-resolved footprinting was used to follow the formation of tertiary structure and protein interactions in the 16S ribosomal RNA (rRNA) during the assembly of 30S ribosomes. As previously observed in much simpler ribozymes, assembly occurs in stages, with individual molecules taking different routes to the final complex. Interactions occur concurrently in all domains of the 16S rRNA, and multistage protection of binding sites of individual proteins suggests that initial encounter complexes between the rRNA and ribosomal proteins are remodeled during assembly. Equilibrium footprinting experiments showed that one primary binding protein was sufficient to stabilize the tertiary structure of the entire 16S 5'-domain. The rich detail available from the footprinting data showed that the secondary assembly protein S16 suppresses non-native structures in the 16S 5'-domain. In doing so, S16 enables a conformational switch distant from its own binding site, which may play a role in establishing interactions with other domains of the 30S subunit. Together, the footprinting results show how protein-induced changes in RNA structure are communicated over long distances, ensuring cooperative assembly of even very large RNA-protein complexes such as the ribosome.

The Dynamic Nature of RNA as Key to Understanding Riboswitch Mechanisms

Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals "on" or "off" for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer-ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) techniques, small-angle X-ray scattering (SAXS) analysis, NMR spectroscopy, and fluorescence spectroscopy, including single-molecule fluorescence resonance energy transfer (smFRET) imaging. One of our research contributions is an approach, termed 2ApFold, that incorporates noninvasive 2-aminopurine modifications in riboswitches. The fluorescence response of these moieties is used to delineate the order of secondary-tertiary structure formation and rearrangements taking place during ligand-induced folding. This information can be used to explore the kinetics of ligand recognition and to analyze the degree of structure preorganization of the free riboswitch state. Furthermore, we discuss a recent smFRET study on the SAM-II riboswitch; this report underscores the importance of choosing strategic labeling patterns that leave maximal conformational freedom to the regulatory interaction. Finally, we comment on how riboswitch ligand recognition appeals to the concepts of conformational selection and induced fit, and on the question of whether riboswitches act under thermodynamic or kinetic control. This Account highlights the fact that a thorough understanding of RNA dynamics in vitro is required to shed light on cellular riboswitch mechanisms. Elucidating these mechanisms will contribute not only to ongoing efforts to target riboswitches with antibiotics but also to attempts to engineer artificial cell regulation systems.