Impact of Reconstituted Cytosol on Protein Stability
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Impact of reconstituted cytosol on protein stability Mohona Sarkara, Austin E. Smitha, and Gary J. Pielaka,b,c,1 Departments of aChemistry and bBiochemistry and Biophysics and cLineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 Edited by Robert L. Baldwin, Stanford University, Stanford, CA, and approved October 15, 2013 (received for review July 27, 2013) Protein stability is usually studied in simple buffered solutions, but physiological mimics because most synthetic polymers are non- most proteins function inside cells, where the heterogeneous globular and relatively inert (15, 16). Protein crowders are better and crowded environment presents a complex, nonideal system. mimics because cells contain large amounts of protein. Protein Proteins are expected to behave differently under cellular crowd- crowders can be destabilizing because they interact with proteins ing owing to two types of contacts: hard-core repulsions and via attractive, weak, nonspecific contacts with the backbone (8, weak, chemical interactions. The effect of hard-core repulsions is 16, 17). Nevertheless, cells are not crowded with one particular purely entropic, resulting in volume exclusion owing to the mere protein. For instance, an E. coli cell possesses 2.4 million protein presence of the crowders. The weak interactions can be repulsive molecules representing ∼4,000 different proteins (18, 19), re- or attractive, thus enhancing or diminishing the excluded volume, sulting in a diverse population of charges, sizes, and amino acid respectively. We used a reductionist approach to assess the effects compositions. of intracellular crowding. Escherichia coli cytoplasm was dia- The intracellular environment is optimal for understanding lyzed, lyophilized, and resuspended at two concentrations. NMR- the physicochemical effects of the cytoplasm, but measuring detected amide proton exchange was then used to quantify the stability in living cells is challenging because application of re- stability of the globular protein chymotrypsin inhibitor 2 (CI2) in ductionist-based methods involving alteration of the cytosol is these crowded solutions. The cytosol destabilizes CI2, and the de- inconsistent with viability (20). Studies of in-cell stability by urea stabilization increases with increasing cytosol concentration. This denaturation suggest that the cytoplasm does not affect stability observation shows that the cytoplasm interacts favorably, but (21, 22), whereas results from McGuffee and Elcock in their nonspecifically, with CI2, and these interactions overcome the sta- pioneering molecular dynamics simulation of the E. coli cyto- bilizing hard-core repulsions. The effects of the cytosol are even plasm (23) point to destabilization. stronger than those of homogeneous protein crowders, reinforc- A nonperturbing method is clearly advantageous for quanti- ing the biological significance of weak, nonspecific interactions. fying the effect of the cellular interior. NMR-detected backbone amide proton exchange (24) is one such method and, unlike acromolecules in Escherichia coli reach concentrations of others, reports stability at the residue level. As discussed below, 300–400 g/L and occupy up to 40% of the cellular volume under certain specific conditions (25) the technique yields the M ðΔ o′ Þ (1), but proteins are normally studied in buffer alone. The effects free energy required to expose amide protons to solvent Gop . of crowding arise from two phenomena, hard-core repulsions For a particular protein, the largest values of this parameter and nonspecific chemical (soft) interactions (2–9). Hard-core occur upon global unfolding and are equivalent to the denatur- Δ o′ repulsions limit the volume available to biological macromole- ation free energy, GD (25). cules for the simple reason that two molecules cannot be in the Detecting resonances from the backbone of globular proteins same place at the same time. This press for space favors com- by NMR in E. coli is difficult because nonspecific interactions pact states over expanded states. The second phenomenon arises limit rotational motion, broadening 15N-1H cross-peaks from because crowders not only exclude volume, but also partici- heteronuclear single quantum coherence (HSQC) spectra into pate in chemical interactions. Even though individually weak, the background (26–29). Here, we use lyophilized E. coli cytosol the high concentration of macromolecules can lead to a large (16, 30–32) to mimic the intracellular environment and quantify net effect. Repulsive nonspecific interactions reinforce the hard- its effects on the stability of the test protein chymotrypsin core repulsions, whereas attractive nonspecific interactions op- inhibitor 2 (CI2), which is amenable to NMR-detected amide pose them. We use the term “nonspecific attractive interactions” proton exchange (33, 34). The cell extracts not only allow us to to distinguish these from specific chemical interactions, such as ligand binding. Significance Our aim is to understand how the crowded and heteroge- neous, intracellular environment affects the equilibrium ther- The cell cytoplasm contains a complex array of macromolecules modynamic stability of globular proteins. Globular proteins are at concentrations exceeding 300 g/L. The natural, most rele- marginally stable in buffer at room temperature (10), with Gibbs “ ” – fi vant state of a biological macromolecule is thus a crowded free energy differences of 5 15 kcal/mol between the ef ciently one. Moving quantitative protein chemistry from dilute solu- packed native (N) state and the ensemble of higher-energy dena- tion to the inside of living cells represents a major frontier that ðΔ o′Þ tured (D) states GD (11). Crowding effects arise from entropic will affect not only our fundamental biological knowledge, but and enthalpic contributions; hard-core repulsions are entropic, fi also efforts to produce and stabilize protein-based pharmaceuti- whereas the consequent nonspeci c chemical interactions are also cals. We show that the bacterial cytosol actually destabilizes our Δ o′ enthalpic. Hard-core repulsions always increase GD for glob- test protein, contradicting most theoretical predictions, but in – ular proteins because D occupies more space than N (12 14). agreement with a novel Escherichia coli model. However, nonspecific interactions can favor N or D, depending fi Δ o′ on their nature (2). Nonspeci c repulsions increase GD because Author contributions: M.S., A.E.S., and G.J.P. designed research; M.S. and A.E.S. per- they make the crowder seem even larger. Nonspecific attractions formed research; M.S., A.E.S., and G.J.P. analyzed data; and M.S., A.E.S., and G.J.P. wrote ′ Δ o the paper. decrease GD because more favorable interactions are able to form as the protein unfolds (2–9). The authors declare no conflict of interest. Most efforts to understand macromolecular crowding use This article is a PNAS Direct Submission. uncharged synthetic polymers as crowding agents (2, 14, 15). 1To whom correspondence should be addressed. E-mail: [email protected]. Such polymers tend to emphasize hard-core repulsions, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. therefore stabilize globular proteins, but they are not accurate 1073/pnas.1312678110/-/DCSupplemental. 19342–19347 | PNAS | November 26, 2013 | vol. 110 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1312678110 Downloaded by guest on September 29, 2021 more directly assess the results of the molecular dynamics sim- of reconstituted cytosol (130.0 g dry weight/L). There was no ulation (23), but also allow the application of a reductionist significant change in CI2 cross-peak intensities over 24 h (SI approach not possible using live cells in that the cytosol con- Appendix,TableS2). These observations indicate that the first centration can be varied. two assumptions are valid; CI2 remained folded, intact, and We extracted the cytoplasm from saturated cultures and re- unaggregated in the reconstituted cytosol for the duration of moved the low-molecular-weight components to observe only the amide proton exchange experiments. the macromolecular effects. After exchanging the labile pro- The third assumption is that kint is rate determining. Un- tons with deuterons, we lyophilized the sample to obtain pow- fortunately, the methods commonly used to verify this assump- dered cytosol. We used NMR-detected amide proton exchange tion (29)—changing the pH and NOESY-detected amide proton (25) to obtain residue level stabilities of 15N-enriched CI2 in exchange (25, 41, 42)—do not work in cytosol. The pH change solutions crowded with 100.0 g dry weight/L and 130.0 g dry method fails because CI2 stability depends on pH (8) and the weight/L reconstituted cytosol. reconstituted cytosol aggregates upon altering the pH. Nor did we observe cross-peaks in a 30 min, time-resolved NOESY- Results detected amide proton exchange spectrum. Increasing the time Suitability of Reconstituted Cytosol. McGuffee and Elcock (23) gave more signal, but the diagonal cross-peaks were lost owing to used an inventory of E. coli proteins compiled by Link et al. (35) the exchange. Instead, we turned to a thermodynamic cycle (43) Δ o′ to design a model cytosol. We made lyophilized cytosol and used to ensure we are measuring Gop (Fig. 1). mass spectrometry and proteomics to identify the proteins. Our The cycle comprises two CI2 variants, I29A;I37H and I57A; list of 233 proteins (SI Appendix, Table S1) contains 33 of the 45 I37H, and two