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Effects of Macromolecular Crowding on the Intrinsically Disordered Proteins c-Fos and p27Kip1 Shannon L. Flaugh† and Kevin J. Lumb*,†,‡ Department of Biochemistry and Molecular Biology and Department of Chemistry, Colorado State University, Fort Collins, Colorado, 80523-1870 Received January 3, 2001
A number of biologically active proteins exhibit intrinsic structural disorder in vitro under thermodynamically ideal conditions. In vivo, however, proteins exist in a crowded, thermodynamically nonideal environment. We tested the hypothesis that intrinsically disordered proteins adopt stable structure under crowded conditions in which excluded volume is predicted to stabilize compact, native conformations. In the presence of macromolecular crowding agents, neither the intrinsically disordered C-terminal activation domain of c-Fos nor the kinase-inhibition domain of p27Kip1 undergoes any significant conformational change that is detected by changes in either circular dichroism or fluorescence spectra. We conclude that molecular crowding effects are not necessarily sufficient to induce ordered structure in intrinsically disordered proteins. Proteins typically require a folded structure that places functional groups in precise positions to participate in molecular recognition and catalysis. It has long been recognized, however, that many functional proteins exhibit biophysical properties expected of unfolded, rather than folded, proteins.1 Such proteins have been variously called natively unfolded,2 intrinsically unstructured,1b or intrinsically disordered.3 An increasing number of intrinsically disordered proteins are being discovered or predicted.1 Some intrinsically disordered proteins have been shown to adopt wellordered structure upon binding other proteins or nucleic acids,1 indicating that organized structure is indeed a functional requirement for certain intrinsically disordered proteins. Proteins studied in vitro are typically in dilute, thermodynamically ideal solutions. However, the cell is a thermodynamically nonideal and crowded environment.4 The effects of macromolecular crowding on chemical activities due to excluded volume are expected to be substantial,5 and experimental studies have shown that macromolecular crowding agents can indeed affect association constants, aggregation and protein folding kinetics.6 One prediction from a theoretical standpoint is that molecular crowding will favor folded structure in proteins as a result of the preferential stabilization of compact states,5 and such effects have been observed experimentally.7 This prediction has potential implications for understanding the properties of intrinsically disordered proteins. Biophysical studies of intrinsically disordered proteins have invariably been performed in vitro in dilute aqueous solution in the absence of the crowding effects that are expected to be important in vivo. A potential outcome of crowding effects is that proteins that are intrinsically disordered in ideal * Corresponding author. Telephone: (970) 491-5440. Fax: (970) 4910494. E-mail:
[email protected]. † Department of Biochemistry and Molecular Biology, Colorado State University. ‡ Department of Chemistry, Colorado State University.
solutions may adopt compact or globular conformations containing secondary structure in a crowded environment. FosAD corresponds to the C-terminal activation domain of human c-Fos (residues 216-310) and is functional for interacting with transcription factors in whole-cell extract.3 p27ID corresponds to the cyclin-dependent kinase inhibition domain of the cell-cycle inhibitor human p27Kip1 (residues 22-97) and is active as a cyclin A-Cdk2 inhibitor.8 Both protein domains are intrinsically disordered as judged by circular dichroism (CD) spectra that are characteristic of unfolded proteins, lack of 1H chemical-shift dispersion and negative 1H-15N nuclear Overhauser effects.3,8 In contrast to the disordered state of the p27 Cdk-inhibition domain in isolation, the crystal structure of p27-inhibited cyclin A-Cdk2 shows that residues 25-93 of the human p27 Cdk-inhibition domain adopt a well-ordered conformation upon binding cyclin A-Cdk2, including helical and strand structure.9 It is currently unknown if FosAD undergoes a conformational change upon binding a target protein, although such behavior has been observed for the intrinsically disordered activation domains of CREB, VP16, and p53.10 Several macromolecules have been used as crowding agents in studies of protein function.6 Here we used three dextrans of average molecular weights 9.5, 37.5, and 77 kDa and Ficoll 70 at concentrations of up to 250 g/L. The concentrations of the dextrans and Ficoll 70 used here cause effects consistent with crowding in other studies6 and are comparable to macromolecule concentrations found in vivo of 200-400 g/L.11 Poly(ethylene glycol) is also an established crowding agent.6a,b However, 5, 15, and 25% (w/v) poly(ethylene glycol) (average molecular mass 3.35 kDa) caused precipitation of p27ID and FosAD. CD spectra reflect secondary structure formation in proteins.12 The CD spectrum of FosAD at 5 °C is reminiscent of an unfolded protein (Figure 1A), in accord with previous results.3 Addition of the helix-stabilizing agent trifluoroethanol (TFE)13 induces changes in the CD spectra that reflect
10.1021/bm015502z CCC: $20.00 © 2001 American Chemical Society Published on Web 03/30/2001
Effects of Macromolecular Crowding
Figure 1. (A) CD spectra of FosAD in 10 mM sodium phosphate (b) and 30% (v/v) TFE (0), the CD spectrum is indicative of helix formation. (B) CD spectra of FosAD in 10 mM sodium phosphate (9) and in the presence of dextrans of average molecular weight 9 kDa (3), 37.5 kDa (4), and 77 kDa (0) at 250 g/L or Ficoll 70 (O) at 250 g/L. Essentially identical spectra were obtained for the dextrans at concentrations of 50, 100, 150, and 200 g/L. (C) Fluorescence spectra of FosAD in 10 mM sodium phosphate (9) and in the presence of dextrans of average molecular weight 9 kDa (3), 37.5 kDa (4), and 77 kDa (0) at 250 g/L or by Ficoll 70 at 250 g/L (O).
formation of helical secondary structure (Figure 1A). In contrast, addition of dextrans and Ficoll 70 did not induce any significant change in the CD spectrum of FosAD above 205 nm that reflects formation of helical or strand secondary structure (Figure 1B). A decrease in the intensity of the CD signal of FosAD at 195 nm was observed in the presence of the dextrans, which may reflect a change in the distribution of the ensemble of disordered conformations or compactness of FosAD. However, interpretation of such changes in CD spectra of unfolded proteins is difficult,12b precluding firm conclusions based on the effects of dextrans on the CD signal at 195 nm.
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Fluorescence spectra reflect structure formation and solvent exposure in proteins, with the emission maximum being the most reliable indicator of conformational change.14 The aromatic fluorescence emission spectrum of FosAD exhibits a maximum at 348 nm (Figure 1C), which is consistent with a solvent-exposed unfolded structure.14 Addition of crowding agents did not cause a change in the emission maximum of Fos AD (Figure 1C). A decrease in emission intensity is observed in the presence of 77 kDa dextran and Ficoll 70 (Figure 1C). The quenching may reflect a change in the ensemble of conformations or compactness of FosAD, although of course trace impurities in these two crowding agents may also induce quenching. The hydrophobic dye ANS (1-anilinonaphthalene-8-sulfonate) provides an empirical probe for the formation of collapsed, partially folded states such as the molten globule.15 Upon binding of molten globules, the intensity of ANS fluorescence increases up to 103 fold and the emission maximum shifts to lower wavelength.15 However, FosAD did not induce a change in the fluorescence emission wavelength or a significant change in the emission intensity (less than approximately 3-fold) of ANS in either the absence or presence of crowding agents (data not shown). p27ID exhibits a higher intrinsic helical propensity than FosAD. While intrinsically disordered at physiological temperatures, marginally stable helix is present in p27ID at 5 °C (Figure 2A).8 Addition of TFE to p27ID induced a marked change in the CD spectrum indicative of a large increase in helix content (Figure 2A). However addition of several crowding agents did not induce any significant change in the CD spectrum of p27ID above 212 nm that reflects formation of helical or strand secondary structure (Figure 2B). Crowding agents did not affect the p27ID fluorescence emission maximum of 350 nm (Figure 2C) or did not affect significantly the fluorescence spectrum of ANS in the presence of p27ID (data not shown). A decrease in the intensity of the CD signal of p27ID at 204 nm was observed in the presence of the 37.5 and 77 kDa dextrans, and the fluorescence emission intensity of p27ID at 350 nm was reduced in the presence of 77 kDa dextran and Ficoll 70 (as observed for FosAD). As noted above, interpretation of such changes is difficult. The spectroscopic data indicate that macromolecular crowding agents do not induce secondary or tertiary structure formation in intrinsically disordered domains from the transcriptional activator c-Fos and the cell-cycle inhibitor p27Kip1. The lack of induced structure is not due to a complete inability of either domain to form secondary structure, since TFE or low temperature (for p27ID) can stabilize helix formation within these two domains. We conclude that, at least for the c-Fos and p27Kip1 domains studied here, macromolecular crowding is not necessarily sufficient to induce structure formation in intrinsically disordered but biologically active proteins. Experimental Section FosAD and p27ID were expressed in Escherichia coli and purified as described previously.3,8 The identities of both
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contained 10 mM FosAD or p27ID in 10 mM sodium phosphate, 1 mM DTT, pH 7.0 plus crowding agents. ANS (Molecular Probes) was used at a concentration of 1 mM. Protein and ANS fluorescence was measured using excitation wavelengths of 280 and 350 nm, respectively. Protein concentrations were determined by absorbance in 6 M GuHCl.3,8 ANS concentration was determined for a stock solution in methanol by absorbance at 372 nm using an extinction coefficient of 7.8 × 103 cm-1 M-1. Acknowledgment. We thank E. A. Bienkiewicz and K. M. Campbell for purified proteins. This work was supported by NIH Grant GM55156 and the Donors of the Petroleum Research Fund, grant 35760-AC4, administered by the American Chemical Society. S.L.F. was supported in part by a Pfizer Summer Undergraduate Research Fellowship. References and Notes
Figure 2. (A) CD spectra of p27ID in 10 mM sodium phosphate (b) and 30% (v/v) TFE (0), the CD spectrum is indicative of helix formation. (B) CD spectra of p27ID in 10 mM sodium phosphate (9) and in the presence of dextrans of average molecular weight 9 kDa (3), 37.5 kDa (4), and 77 kDa (0) at 250 g/L or Ficoll 70 (O) at 250 g/L. Essentially identical spectra were obtained for the dextrans at concentrations of 50, 100, 150, and 200 g/L. (C) Fluorescence spectra of p27ID in 10 mM sodium phosphate (9) and in the presence of dextrans of average molecular weight 9 kDa (3), 37.5 kDa (4), and 77 kDa (0) at 250 g/L or by Ficoll 70 at 250 g/L (O).
proteins were confirmed with electrospray mass spectrometry, with the expected and observed masses agreeing to within 1 Da. Denaturing (SDS) polyacrylamide electrophoresis indicated that both proteins were essentially free of contaminating proteins. Dextrans of average molecular weight 9.5, 37.5, and 77 kDa (Sigma) were used at concentrations of 50, 100, 150, 200, and 250 g/L. Ficoll 70 (Fluka) was used at a concentration of 250 g/L. CD spectra were collected at 5 °C with a Jasco J720 spectrometer. Samples contained 20 mM FosAD or p27ID in 10 mM sodium phosphate and 1 mM dithiothreitol (DTT), pH 7.0, plus crowding agents. Fluorescence spectra were collected at 5 °C with an Aviv ATF105 fluorometer. Samples
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