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Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

ABSTRACT: Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg2+ concentrations typical of the cell's interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl2 and polyethylene glycol (PEG) concentrations. The resulting phase diagrams show that perturbations to folding by each variable do not overlap. A favorable enthalpy change drives the formation of compact, native-like structures, but requires Mg2+ ions at all temperatures studied (5-55°C). PEG reduces the entropic cost of helix assembly and increases correlations between RNA segments at all temperatures. The phase diagrams also revealed a semi-compact intermediate between the unfolded and folded ensemble that is locally more flexible than the unfolded state, as judged by SHAPE modification. These results suggest that environmental variables such as temperature and solute density will favor different types of RNA structures.

SUBMITTER: Kilburn D

PROVIDER: S-EPMC5100557 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

Kilburn Duncan D Behrouzi Reza R Lee Hui-Ting HT Sarkar Krishnarjun K Briber Robert M RM Woodson Sarah A SA

Nucleic acids research 20160704 19

Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg<sup>2+</sup> concentrations typical of the cell's interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different ...[more]

PMID: 27378777

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Project description:The osmolyte trimethylamine N-oxide (TMAO) accumulates in the cell in response to osmotic stress and increases the thermodynamic stability of folded proteins. To understand the mechanism of TMAO induced stabilization of folded protein states, we systematically investigated the action of TMAO on several model dipeptides (leucine, L(2), serine, S(2), glutamine, Q(2), lysine, K(2), and glycine, G(2)) in order to elucidate the effect of residue-specific TMAO interactions on small fragments of solvent-exposed conformations of the denatured states of proteins. We find that TMAO preferentially hydrogen bonds with the exposed dipeptide backbone but generally not with nonpolar or polar side chains. However, interactions with the positively charged Lys are substantially greater than with the backbone. The dipeptide G(2) is a useful model of the pure amide backbone; interacts with TMAO by forming a hydrogen bond between the amide nitrogen and the oxygen in TMAO. In contrast, TMAO is depleted from the protein backbone in the hexapeptide G(6), which shows that the length of the polypeptide chain is relevant in aqueous TMAO solutions. These simulations lead to the hypothesis that TMAO-induced stabilization of proteins and peptides is a consequence of depletion of the solute from the protein surface provided intramolecular interactions are more favorable than those between TMAO and the backbone. To test our hypothesis, we performed additional simulations of the action of TMAO on an intrinsically disordered A?(16-22) (KLVFFAE) monomer. In the absence of TMAO, A?(16-22) is a disordered random coil. However, in aqueous TMAO solution, A?(16-22) monomer samples compact conformations. A transition from random coil to ?-helical secondary structure is observed at high TMAO concentrations. The coil to ?-helix transition is highly cooperative especially considering the small number of residues in A?(16-22). Our work highlights the potential similarities between the action of TMAO on long polypeptide chains and entropic stabilization of proteins in a crowded environment due to excluded volume interactions. In this sense, the chemical chaperone TMAO is a nanocrowding particle.

Project description:Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.

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Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

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Entropic stabilization of folded RNA in crowded solutions measured by SAXS.

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