Denaturation of RNA Secondary and Tertiary Structure by Urea: Simple Unfolded State Models and Free Energy Parameters Account for Measured <i>m</i>-Values

Lambert, Donald H.; Draper, David E.

doi:10.1021/bi301103j

Cited by 55 publications

(110 citation statements)

References 66 publications

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“…An example is the mechanism of open complex formation and stabilization in transcription initiation by Escherichia coli RNA polymerase (RNAP) at a DNA promoter (47). Here, information about interactions of solutes and noncoulombic (Hofmeister) interactions of salt ions with nucleic acid functional groups is also needed; this has been obtained for urea (11,12), and research with other solutes and salts is in progress. The small effects of urea and Hofmeister salts on the rate and equilibrium constants for the step involving opening of 13 bp of duplex promoter DNA provide evidence that RNAP opens this region in the active site cleft (14,48,49).…”

Section: Discussionmentioning

confidence: 99%

“…The large effects of urea, glycine betaine, and different Hofmeister salts on the subsequent step(s) that stabilize the initial open complex provide evidence for large-scale coupled folding and assembly of mobile elements of RNAP to form a clamp on the downstream duplex DNA (14,(48)(49)(50). Solutes are also excellent probes of intermediates and TS for RNA folding, because burial of base ASA is disfavored by all solutes whereas burial of backbone ASA is favored by some solutes (12,13 (Table S5). Free energies are arbitrarily chosen; only the ASA scale is quantitative.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the interactions of key denaturants, osmolytes, and Hofmeister salts with the functional groups of proteins and nucleic acid have been determined, allowing these solutes to be used as thermodynamic and kinetic-mechanistic probes of the amount and the composition of the surface buried or exposed in the steps of a noncovalent protein or nucleic acid process like helix formation, folding, or binding (6)(7)(8)(9)(10)(11)(12)(13)(14). For protein folding, urea and guanidinium chloride (GuHCl) thermodynamic m-values (derivatives of the standard free energy of folding with respect to denaturant concentration) are determined almost entirely by the preferential interactions of these denaturants with the hydrocarbon and amide surface buried in folding (6):…”

Section: Denaturant Probes Of Changes In Amide and Hydrocarbon Surfacmentioning

confidence: 99%

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Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Guinn¹,

Kontur

Tsodikov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.etermination of the mechanism of protein folding [unfolded (U) → folded (F)] is a long-standing goal of biophysical research. Folding of a single domain globular protein is a very highly cooperative (thermodynamically two-state) process. From analysis of folding kinetic data for CI2 and barnase, Fersht et al.(1) concluded that proteins fold through a high-free-energy transition state (TS) with partially formed elements of native structure. Recently, Barrick and Sosnick (2) concluded that an ensemble of unstable, rapidly reversible intermediates form early in the folding mechanism, and that the most advanced and unstable of these undergoes a rate-determining conformational change with transition state TS. This transit step, slower than the reverse direction of previous rapidly reversible steps, is followed by rapid propagation of folding. Most proposals for the initial intermediates invoke unstable regions of α-helix and/or β-sheet; these are thought to coalesce and/or rearrange to form TS. Kay and coworkers (3) characterized a marginally stable early intermediate of Fyn SH3 domain and found that interactions of amide groups formed earlier in the folding pathway than interactions of methyl groups, indicating that 2°structure formed before the 3°fold. Recent hydrogen exchange pulse-labeling experiments analyzed with mass spectrometry by Englander, Marqusee, and collaborators (4) indicate that folding of RNase H occurs t...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Denaturant Probes Of Changes In Amide and Hydrocarbon Surfacmentioning

confidence: 99%

See 1 more Smart Citation

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Guinn¹,

Kontur

Tsodikov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Effects of cosolutes on RNA folding were not significantly investigated until the last decade. Studies on RNAs with either secondary and/or tertiary structures report that cosolutes such as betaine, proline, and methanol, almost always destabilize secondary structures, while having mixed effects on tertiary structure (Lambert & Draper, 2007; Lambert & Draper, 2012; Lambert et al, 2010; Soto et al, 2007). Several osmolytes have been shown to interact with the nucleobase, sugar, and phosphate of RNAs, with examples of both favorable and unfavorable interactions (Lambert & Draper, 2007).…”

Section: Bridging the Gap Between In Vitro And In Vivo Rna Foldingmentioning

confidence: 99%

Bridging the gap betweenin vitroandin vivoRNA folding

Leamy

Assmann

Mathews

et al. 2016

Quart. Rev. Biophys.

122

132

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Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

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“…Experimental studies have shown that RNA undergoes denaturation in the presence of urea, and such studies are useful in monitoring the unfolding pathways and the associated thermodynamic changes. 3–7 Chemical denaturants such as urea, guanidinium chloride, and alcohols have been commonly used to elucidate protein folding pathways and also to probe the intramolecular interactions that stabilize the native state of proteins. 8–17 While the molecular mechanism by which urea destabilizes proteins has been a subject of several investigations, how urea facilitates RNA unfolding is not well understood.…”

Section: Introductionmentioning

confidence: 99%

Dispersion Interactions between Urea and Nucleobases Contribute to the Destabilization of RNA by Urea in Aqueous Solution

et al. 2015

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Urea has long been used to investigate protein folding and, more recently, RNA folding. Studies have proposed that urea denatures RNA by participating in stacking interactions and hydrogen bonds with nucleic acid bases. In this study, the ability of urea to form unconventional stacking interactions with RNA bases is investigated using ab initio calculations (RI-MP2 and CCSD(T) methods with the aug-cc-pVDZ basis set). A total of 29 stable nucleobase-urea stacked complexes are identified in which the intermolecular interaction energies (up to −14 kcal/mol) are dominated by dispersion effects. Natural bond orbital (NBO) and atoms in molecules (AIM) calculations further confirm strong interactions between urea and nucleobases. Calculations on model systems with multiple urea and water molecules interacting with a guanine base lead to a hypothesis that urea molecules along with water are able to form cage-like structures capable of trapping nucleic acid bases in extrahelical states by forming both hydrogen bonded and dispersion interactions, thereby contributing to the unfolding of RNA in the presence of urea in aqueous solution.

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Denaturation of RNA Secondary and Tertiary Structure by Urea: Simple Unfolded State Models and Free Energy Parameters Account for Measured m-Values

Cited by 55 publications

References 66 publications

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Bridging the gap betweenin vitroandin vivoRNA folding

Dispersion Interactions between Urea and Nucleobases Contribute to the Destabilization of RNA by Urea in Aqueous Solution

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