Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins

Gao, Jianmin; Bosco, Daryl A.; Powers, Evan T.; Kelly, Jeffery W.

doi:10.1038/nsmb.1610

Cited by 196 publications

(200 citation statements)

References 61 publications

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“…Most simply, they asked whether a mutation that affects the propensity of a sequence to form an alpha helix, say decreasing this propensity, destabilizes overall folding of the protein by the same amount. This prediction bore out for mutations in exposed residues on the protein, but this simple rule is not followed for a buried side chain, as desolvation and interactions with neighboring residues affect the energetic contribution of these sides chains to folding (51,(64)(65)(66). As noted above, this approach and potential modularity may be more applicable to RNA tertiary structures given their limited packing interfaces and the sparse distribution of tertiary contacts throughout a structure.…”

Section: Discussionmentioning

confidence: 99%

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Bisaria

Greenfeld

Limouse

et al. 2017

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

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Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔG align , the probability of aligning tertiary contact partners, and ΔG tert , the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔG HJH ) or from changes in the electrostatic environment (ΔG +/− ) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔG tert ). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔG tert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.RNA folding | single-molecule FRET | RNA tertiary structure | folding kinetics | folding thermodynamics S tructured RNAs are integral to many biological processes, including translation, genome maintenance, and the regulation of gene expression (1, 2). These processes require RNA to fold into intricate 3D structures and to undergo a series of structural transitions (3, 4). As such, an important goal has been to characterize these states, in terms of their conformations and the rates and equilibria that describe the transitions between them (5-8). A more distant but ultimately far-reaching challenge is to quantitatively predict the kinetics and thermodynamics of RNA transitions, an accomplishment that would demonstrate fundamental understanding and effectuate engineering of these systems.Quantitative analyses of the melting temperatures for nucleic acid duplexes led to predictive rules for RNA secondary structure stability, known as nearest neighbor or Turner rules, such that the energetics of helix formation can be predicted by considering only the identity of each base pair, the identity of its immediate neighbors, and the salt concentration (9-11). In this model, the energetic contribution of each base pair step is additive and can be summed to predict the free energy to a...

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Section: Discussionmentioning

confidence: 99%

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Bisaria

Greenfeld

Limouse

et al. 2017

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

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“…It has been shown that electrostatic screening is the main reason for the distinct backbone conformational preferences of amino acid residues in peptides and proteins (53), the nearest neighbor effect (54), and the formation of transient β-strands in unfolded proteins (55). It has also been shown, using amide to ester mutations, that H bonds in the protein interior are stronger than those exposed to solvent (56)(57)(58).…”

Section: Ir Spectroscopy Of Water Molecules In the Neighborhood Of Pumentioning

confidence: 99%

Origin of hydrophobicity and enhanced water hydrogen bond strength near purely hydrophobic solutes

Grdadolnik

Merzel

Avbelj

2016

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

201

145

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Hydrophobicity plays an important role in numerous physicochemical processes from the process of dissolution in water to protein folding, but its origin at the fundamental level is still unclear. The classical view of hydrophobic hydration is that, in the presence of a hydrophobic solute, water forms transient microscopic "icebergs" arising from strengthened water hydrogen bonding, but there is no experimental evidence for enhanced hydrogen bonding and/or icebergs in such solutions. Here, we have used the redshifts and line shapes of the isotopically decoupled IR oxygen-deuterium (O-D) stretching mode of HDO water near small purely hydrophobic solutes (methane, ethane, krypton, and xenon) to study hydrophobicity at the most fundamental level. We present unequivocal and model-free experimental proof for the presence of strengthened water hydrogen bonds near four hydrophobic solutes, matching those in ice and clathrates. The water molecules involved in the enhanced hydrogen bonds display extensive structural ordering resembling that in clathrates. The number of ice-like hydrogen bonds is 10-15 per methane molecule. Ab initio molecular dynamics simulations have confirmed that water molecules in the vicinity of methane form stronger, more numerous, and more tetrahedrally oriented hydrogen bonds than those in bulk water and that their mobility is restricted. We show the absence of intercalating water molecules that cause the electrostatic screening (shielding) of hydrogen bonds in bulk water as the critical element for the enhanced hydrogen bonding around a hydrophobic solute. Our results confirm the classical view of hydrophobic hydration.hydrophobic hydration | hydrogen bonding | IR spectroscopy | electrostatic screening | ab initio molecular dynamics D espite its great importance in numerous phenomena, the origin of hydrophobicity remains one of the most disputed topics in science (1-5). Experimental studies have shown that small purely hydrophobic solutes (alkanes and noble gases) in water increase the order (6, 7) and restrict the mobility (8) of neighboring water molecules. There are several opposing views on how to explain these data. The classical view is that a solute modifies water structure by forming transient, semiordered clathrate-like clusters ("icebergs"; used here only as a loose term) around it, arising from enhanced water hydrogen bonding (H bonding) (6, 7). This enhancement is brought about by either strengthening (9) or increasing the number of water to water H bonds (10). The classical view explains the characteristic changes in the thermodynamic variables of hydrophobic hydration (positive ΔG, ΔC p , negative ΔS, and ΔH) and the restricted mobility of water molecules observed by NMR (8). Neutron diffraction (11, 12) and extended X-ray absorption fine structure (EXAFS) (13) studies, however, show that the water molecules around small purely hydrophobic molecules do not differ significantly from those in pure liquid water. According to the dynamic view, the hydrophobic solute causes slowdown of t...

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“…6B). The hydrogen bond interactions between the PH domain and EF hand residues involve residues whose side chain donor and acceptor atoms are 3-4 Å apart, significantly weaker in energy than the average hydrogen bond lengths observed in proteins (ϳ1.5-2 Å) (35). Taken together with the absence of electrostatic interactions or water bridges at this interface, we hypothesized that the PH domain is mobile in solution, existing in equilibrium between the conformation observed in crystal structures and an "open" conformation where the PH domain moves so that the residues required for G␤␥ binding become accessible.…”

Section: Rac1 and G␤␥ Bind Distinct Surfaces On Plc-␤-plc-␤2mentioning

confidence: 99%

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site

Kadamur

Ross

2016

Journal of Biological Chemistry

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Mammalian phospholipase C-␤ (PLC-␤) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how G␣ q and Rac1 bind PLC-␤, there is a lack of consensus regarding the G␤␥ binding site in PLC-␤. Using FRET between cerulean fluorescent protein-labeled G␤␥ and the Alexa Fluor 594-labeled PLC-␤ pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal G␤␥ binding region in PLC-␤3. We show that the isolated PH domain can compete with full-length PLC-␤3 for binding G␤␥ but not G␣ q , Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-␤ PH domain as the G␤␥ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-␤, necessitating conformational rearrangement to allow G␤␥ binding. Blocking PH domain motion in PLC-␤ by crosslinking it to the EF hand domain inhibits stimulation by G␤␥ without altering basal activity or G␣ q response. The fraction of PLC-␤ cross-linked is proportional to the fractional loss of G␤␥ response. Cross-linked PLC-␤ does not bind G␤␥ in a FRETbased G␤␥-PLC-␤ binding assay. We propose that unliganded PLC-␤ exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-␤ modulates G␤␥ access to its binding site. Phospholipase C (PLC)2 isozymes integrate signaling inputs downstream of diverse receptors to catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the inner leaflet of the plasma membrane. This reaction generates two products, inositol 1,4,5-trisphosphate and diacylglycerol, important second messengers and second messenger precursors that regulate multiple cellular processes (1). The mammalian genome encodes six families of PLCs (␤, ␦, ␥, ⑀, , and ) that share a conserved core architecture composed of a pleckstrin homology (PH) domain, four EF hands, a split TIM barrel, and a C2 domain. The TIM barrel contains the active site, and its two halves are separated by the so-called X-Y linker that is thought to occlude the active site and whose motion is central to the regulation of activity. In the PLC-␤ isozymes, the linker is highly cationic, flanked by a highly anionic region, and disordered. Sondek and co-workers (2) speculated that PLC recruitment to the plasma membrane by protein activators causes electrostatic repulsion of the linker by negatively charged phospholipids that moves it away from the active site to relieve autoinhibition. Proteolysis or genetic removal of the linker elevates basal enzyme activity across many PLC isozymes (3, 4), supporting the generality of this mechanism. However, mutant PLC-␤s that lack this linker are activated further by their physiological ligands (2), suggesting the existence of additional regulatory mechanisms that are yet to be discovered.The four mammalian PLC-␤ ...

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Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins

Cited by 196 publications

References 61 publications

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Origin of hydrophobicity and enhanced water hydrogen bond strength near purely hydrophobic solutes

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site

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