Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

167

195

Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer's disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a "superposition of ensembles" hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.protein folding | intrinsically disordered proteins | molecular dynamics simulations | osmolytes | tau protein M ost proteins in the human body have a well-defined, stable three-dimensional structure that is intimately tied to their physiological function. In the past few decades however, researchers have also identified a class of proteins that are natively unstructured. The latter, often referred to as intrinsically disordered proteins (IDPs) (1), are widespread and have the ability to quickly change their conformations to participate in a variety of biological processes. IDPs typically contain multiple charged side chains and few hydrophobic residues. Despite these characteristics, IDPs are not typically found in extended states but rather populate compact states due to hydrogen bonds and salt bridges (2, 3). IDP structures are highly regulated in the cell, and aberrant regulation often results in protein aggregation.In this paper we consider the effect of external agents, specifically osmolytes, in regulating IDP structure and aggregation properties. To carry out this study, we focused on the microtubule-associated protein tau, a soluble (4), archetypical IDP found in the nervous system that helps regulate and stabilize microtubules (5, 6). When the regulation of tau structure and activity is compromised, tau loses its ability to bind to microtubules, and disassociated tau proteins can aggregate into supramolecular assemblies with a cross-β structure (7-9) typical of amyloid fibers. This aggregation process is a pathological hallmark of Alzheimer's disease and other forms of dementia known as tauopathies (10, 11). We consider here a segment of tau, the R2/wt fragment 273 GKVQIINKKLDL 284 , which contains the highly aggregation ...

Section: Resultsmentioning

confidence: 99%

Regulation and aggregation of intrinsically disordered peptides

Levine¹,

Larini²,

LaPointe

et al. 2015

167

195

“…However, TMAO molecules are found to be excluded from various protein backbone and sidechain units, thus destabilizing the unfolded state (59). Moreover, many studies have shown that both TMAO and urea can alter the structure and dynamics of the H-bonding network of water, thus resulting in a change in protein stability (69).…”

Section: Discussionmentioning

confidence: 99%

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Pazos

Gai

2014

227

237

Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.N ature employs a variety of small organic molecules to cope with osmotic stress. Trimethylamine N-oxide (TMAO) is one such naturally occurring osmolyte that protects intracellular components against disruptive stress conditions (1). In particular, previous studies have shown that TMAO is able to enhance protein stability and to counteract the denaturing effect of urea (2, 3). TMAO (Fig. 1, Inset) adopts a skewed tetrahedral structure with a charged oxygen capable of accepting hydrogen bonds (H bonds) and three hydrophobic (methyl) groups. This amphiphilic structural arrangement makes TMAO a rather special cosolvent, because it can form H bonds with water, self-associate in a manner similar to surfactants, and show preferential interactions with or exclusion (4-12) from certain protein functional groups (13-23). Indeed, these molecular properties of TMAO have been used, either individually or in combination, to rationalize its biological activities. For example, a prevailing view about TMAO is that its osmotic and protective role is caused by the molecule's tendency to be preferentially depleted from protein surfaces, as suggested by physicochemical measurements (24-28). This thermodynamic picture, as described by Bolen and coworkers (29), implies that the protein is preferentially hydrated. Although this notion is consistent with TMAO's being a protecting osmolyte, to the best of our knowledge no experimental studies have been carried out to directly examine the effect of TMAO on protein hydration dynamics, an aspect that is also important to protein function. Herein, we use twodimensional infrared (2D IR) spectroscopy and a site-specific IR probe to explore this critical issue and gain insight toward achieving a microscopic understanding of the molecular mechanism of the protecting action of TMAO.2D IR spectroscopy is capable of assessing the frequencyfrequency correlation function (FFCF) of a given IR probe, which reports on the underlying dynamics of events that lead to fluctuations of its vibrational frequency (30). For an IR probe that is able ...

“…Solvation studies with model compounds have suggested that TMAO is excluded from unfolded proteins because of its very unfavorable interactions with the peptide backbone (9, 10). Several molecular dynamics (MD) simulation studies have been consistent with this conclusion (11,12). However, they have proposed a wide array of explanations for this preferential hydration ranging from unfavorable electrostatic interactions (13,14) to "nanocrowder" effects (15,16).…”

mentioning

confidence: 84%

Trimethylamine N -oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine

Liao

Manson

DeLyser

et al. 2017

158

197

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air-water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer-water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins.osmolytes | protein folding | mechanism | spectroscopy | MD simulations M any organisms use small organic osmolytes to stabilize proteins in harsh environments, such as when the salinity is highly variable (1). In particular, trimethylamine N-oxide (TMAO) is known to counteract the denaturing effects of urea as well as salts, and it is present at high concentrations in some aquatic organisms (2). Its effects are often compared with the ions on the left side of the Hofmeister series, which help stabilize the native, folded structures of proteins (Fig. 1).Because of their fundamental biophysical importance, many studies have investigated the behavior and effects of osmolytes. In particular, Timasheff and coworkers (3, 4) proposed that osmolyte effects result from the relative partitioning of these molecules between the bulk solution and the protein-water interface. Stabilization should occur when osmolytes are depleted from the protein-water interface, but proteins will unfold when osmolytes accumulate at this interface. Accordingly, osmolyte effects are often interpreted in terms of an effective protein-water "surface tension." In fact, despite the significant differences between protein surfaces and air-water interfaces, osmolyte effects are often, although not always, consistent with their effect on the air-water interfa...