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Osmolytes and protein-protein interactions Amy E Rydeen, Eric M. Brustad, and Gary J. Pielak J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03903 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018
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Journal of the American Chemical Society
Osmolytes and protein-protein interactions a
a,*
Amy E. Rydeen , Eric M. Brustad , and Gary J. Pielak a
a,b,c,d,*
b
c
Department of Chemistry, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
d
Supporting Information Placeholder
ABSTRACT: Cells survive fluctuations in osmolality by accumulating and depleting highly soluble, usually neutral, small organic compounds. Natural selection has converged on a small set of such molecules, called osmolytes. The biophysical characterization of osmolytes, with respect to proteins, has centered on tertiary structure stability. Data about their effect on protein assemblies, whose formation is driven by surface interactions, is lacking. Here, we investigate the effects of osmolytes and related molecules on the stabilities of a protein and a protein complex. The results demonstrate that osmolytes are not differentiated from other cosolutes by their stabilizing influences on protein tertiary structure but by their compatibility with the interactions between protein surfaces that organize the cellular interior.
Recent studies have renewed interest in the physiological relevance of macromolecular surfaces, specifically the importance of electrostatic interactions. The intracellular consequence of perturbing surface electrostatics is highlighted, firstly, by the formation of stress granules, aggregated RNA and RNA-binding proteins.1 This subset of ribonucleoprotein granules are thought to form in response to osmotic stress-induced screening of repulsive surface electrostatic interactions and subsequent aggregation of likecharged macromolecules, such as mRNA.1-2 Secondly, in vivo, charged residues on protein surfaces interact with neighboring cytoplasmic components, altering protein stability.3-6 Coupled with the sensitivity of nucleic acids, proteins, protein-nucleic acid and protein-membrane interactions to changes in ion concentration,7-13 these findings cultivate the emerging view of a cytoplasm that is governed by transient,
weak, charge-mediated interactions between biomolecular surfaces.14-16 Herein, we demonstrate that compatibility with interactions between charged surfaces, specifically protein subunits, determines the molecules employed to cope with cellular stress. Environmental stresses, including fluctuations in salinity, extracellular freezing and desiccation, drive water from the cell thereby increasing the concentration of intracellular solutes, including ions.17-18 Organisms, such as Escherichia coli, initially respond to osmotic stress by the uptake of inorganic ions, which partially restore cytoplasmic water concentration and growth rates.19 However, cellular survival during prolonged exposure to osmotic stress requires a mechanism to reestablish cellular homeostasis. Natural selection arrived at a simple solution, the accumulation of small organic molecules called osmolytes.8 The molecules nature chose to serve as osmolytes vary across the domains of life, but molecules compatible with macromolecular structure and function are preferred.20 To date, the biophysical characterization of compatible osmolytes has centered on their effects on protein tertiary structure and stability.21-24 Osmolytes influence protein folds through a mechanism that targets the universal component of protein, the amide backbone, although the mechanism remains contraversial.25-26 Stabilizing osmolytes are preferentially excluded from protein surfaces, driven by a thermodynamic distaste for the protein backbone.27 Urea, the denaturing osmolyte, destabilizes protein structure via favorable interactions with the backbone.28 Consequently, osmolytes have been characterized as molecules that affect the equilibrium between a protein’s folded state and unfolded ensemble (i.e.
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protein stability).29 This depiction is incomplete because it omits protein-protein interactions; a ubiquitous feature in the maintenance of cellular homeostasis.30 Current theoretical models that predict osmolyte effects on protein folds, do not account for osmolyte-induced changes in surface interactions between folded proteins.31-33 Furthermore, there is a paucity of data about the influence of osmolytes on protein-protein interactions.31-38 In general, osmolytes that stabilize folds also stabilize protein complexes but there are exceptions. For these reasons, a consensus on osmolyte effects on protein-protein interactions has yet to be reached.33 Here, we evaluate the effect of a panel of osmolytes and related non-osmolytes on the stabilities of a model protein and a protein complex to provide a more comprehensive view of the advantageous features of osmolytes. We also hope that directly contrasting osmolytes and non-osmolytes will contextualize past conflicting results that prevented a unified view of osmolytes and protein-protein interactions. Our panel of solutes (Figure S1) includes 14 osmolytes, that encompass the chemical diversity found in various organisms and eight molecules that are critical for cellular function but are not osmolytes, including amino acids (arginine, glutamine, and lysine), prominent intracellular cations (polyamines) and anions (acetate and citrate). Solubility is a key determinate of the molecules that accumulate at high concentrations in cells.39 Consequently, we excluded other uncharged metabolites because of their poor solubility.40
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both acidic with pI values of 4.6 and 4.5, respectively, which is representative of the majority of proteins in E. coli and Bacillus subtilis.47-48 °+ ) and The free energies of SH3 unfolding (∆𝐺'→) °+ GB1 dimer dissociation (∆𝐺"→, ) were determined by using 19F NMR facilitated by the genetic incorporation of the unnatural amino acid, 4-(trifluoromethyl)-LPhenylalanine (TFMF, Figure 1A Inset).49-50 For SH3, TFMF was introduced in place of Tyr37 (Figure S2A), which is buried in the folded protein. TFMF minimally impacts SH3 stability, reducing the midpoint temperature of denaturation in buffer from 36 °C to 32 °C (Figure S3). The 19F NMR spectra of SH3Y37TFMF exhibit two resonances, one from the folded state at -61.96 ppm and the other from the unfolded ensemble at -61.80 ppm, (Figure 1A).
Wild-type GB1 is monomeric, but a single mutation, A34F, induces a side-by-side symmetric homodimer.51-52 Incorporating TFMF in place of A34 (Figure S2B) induces dimerization in a similar fashion to A34F and provides 19F spectra exhibiting two concentration-dependent resonances (Figure 1B) indicative of the monomer (-61.90 ppm) and dimer (-62.05 ppm) populations. 15N-1H heteronuclear single quantum correlation (HSQC) spectra of the GB1-A34TFMF closely match those of GB1-A34F (Figure S4) suggesting the structure is maintained. Small changes in shifts are observed for residues lining the hydrophobic dimer interface and these disruptions are reflected by a 𝐾" (440 ± 10 𝜇M) 7.5 times greater than the A34F variant.52
As a model protein, we selected the SH3 domain of the Drosophila signal transduction protein drk. SH3 is a model globular protein, well suited for protein folding studies, because its thermal denaturation is reversible, two state and the folded and unfolded populations are in a dynamic equilibrium.5, 41 Importantly, SH3 is destabilized in cells in response to osmotic stress, and the destabilization is alleviated by adding the osmolyte betaine.5, 42 As a model protein complex, we chose the engineered side-by-side symmetric homodimer of the B1 domain of the streptococcal immunoglobulin binding protein G (GB1). The dimer interface is composed of hydrogen bonded anti-parallel beta-sheets and a hydrophobic interaction, both of which are common structural motifs in naturally occurring protein interfaces.43-44 The dissociation constant, 𝐾" , of the GB1 dimer, >1 𝜇M, is in the range of the transient protein-protein interactions that organize and regulate cells.3, 45 Furthermore, the interface of the dimer is the same one used in the natural interaction of GB1 with IgG.46 SH3 and GB1 are
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Journal of the American Chemical Society Figure 1. Quantification of protein- and dimer-stabil19 ity by F NMR (298 K, pH 7.4). (A) TFMF-labeled SH3 displays resonances corresponding to the folded state (F) and the unfolded ensemble (U). Inset: Structure of 4-(trifluoromethyl)-L-Phenylalanine (TFMF). (B) TFMF-labeled GB1 shows concentration-dependent dimer (D) and monomer (M) resonances.
Cosolute-induced changes in the stability of SH3Y37TFMF and the GB1-A34TFMF dimer, with respect °+ °+ to buffer, ∆∆𝐺'→) and ∆∆𝐺"→, (Figure 2, Table S1), are defined such that increased stability gives a posi°+ was detertive value. For GB1-A34TFMF, ∆∆𝐺"→, mined at a single concentration, 125 𝜇M. For a subset °+ of cosolutes, ∆∆𝐺"→, values were determined from binding isotherms, and the results agree with single concentration measurements. (Figure S5 and Table S2). Osmolytes and non-osmolytes are not distinguishable in terms of their effects on SH3 stability. All cosolutes tested are stabilizing, with the exception for the denaturing osmolyte urea (Figure 2A). Quantification °’
of 𝛥𝛥𝐺'→) in citrate was not possible due to the absence of a resonance corresponding to the unfolded ensemble and the value is reported as greater than the most stabilizing quantifiable cosolute, lysine. Interestingly, non-osmolytes (citrate, lysine, and acetate) provide the largest enhancements in SH3 stability.
cond, formation of the GB1 dimer is driven by a hydrophobic interaction that overcomes self-repulsion between two like-charged monomers.51 To investigate the extent to which charge-charge interactions dictate GB1 dimerization, 19F spectra were acquired in buffer between pH 7 and pH 4. A shift from predominately monomeric- to predominately dimeric- GB1 is observed with decreasing pH (Figure S6). In addition, a small amount of a higher-order aggregate, as characterized by dynamic light scattering (Figure S7), forms at pH values less than 6. Furthermore, the population of dimer correlates positively with the concentration of NaCl (Figure S8). These data demonstrate that GB1 dimer stability is influenced by electrostatic interactions and that Coulombic screening of repulsive charges promotes association. We therefore evaluated the effect of a subset of cosolutes, both osmolytes and non-osmolytes, at pH 4 where electrostatic repulsion would be minimized due to neutralization of acidic surface sidechains. At this pH, the population of dimer was unaffected by adding cosolutes, suggesting that in absence of charge-charge subunit repulsion, the cosolutes have a negligible influence on the equilibrium between dimeric and monomeric GB1 (Figure S9 and Table S3).
In contrast, a distinction is evident between osmo°’
lyte and non-osmolyte GB1 𝜟𝜟𝑮𝑫→𝑴 values (Figure 2B). With the exception of glycine and glutamate, osmolytes minimally perturbed dimer stability, increas°’
ing 𝜟𝜟𝑮𝑫→𝑴 by less than 1 kcal/mol. All non-osmolytes tested showed a more pronounced (1 – 2 °’
kcal/mol) change in 𝜟𝜟𝑮𝑫→𝑴 in favor of dimer formation. Stabilization of the GB1 dimer trends with the absolute charge of the cosolute, and species with the highest overall charge induced the largest dimer stabilization. The stabilization of the GB1 dimer in the presence of charged cosolutes could result from disruptions of the electrostatic interactions between GB1 subunits. This idea is corroborated by several observations. First, the stability of monomeric GB1 decreases concomitant with decreasing pH in living E. coli cells53 because as the pH drops, the surface of cytosolic E. coli macromolecules become more cationic generating an increased number of favorable electrostatic interactions with polyanionic GB1 (-4 at pH 7.4).16 Se-
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urea NaCl tmao lysine citrate taurinealanine proline acetate arginine glycine betaine sorbitol sucrose glycerol ?-alanine spermine trehalose sarcosine glutamate glutamine* spermidine
°+ Figure 2. Changes in free energy (∆∆𝐺 °+ = ∆𝐺67879:;< − °’
°+ ) for SH3 unfolding (𝛥𝛥𝐺'→) , panel A) and GB1 ∆𝐺>:??
