Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (>400 g͞liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions. M ost proteins require a defined three-dimensional structure to perform their function. Intrinsically disordered proteins seem paradoxical because they lack stable structure, yet they play key roles in diverse biological processes including signal transduction, transcription, and neurodegenerative diseases (1, 2). Here, we report results from studies of the so-called intrinsically disordered protein, FlgM, a 97-residue polypeptide from Salmonella typhimurium that regulates flagellar synthesis by binding the transcription factor , as shown by the disappearance of crosspeaks from residues in the C-terminal half of FlgM in the FlgM-28 complex (8). This bipartite behavior (i.e., disappearance of crosspeaks from the C-terminal half with retention of crosspeaks from the Nterminal half) provides a valuable built-in control for studying the response of FlgM to different solution conditions. We discuss our results in terms of two types of intrinsically disordered proteins: those that gain structure under crowded conditions and those that do not. FlgM is an example of both types in a single protein. Materials and MethodsFlgM was overexpressed and purified as described (4,8). In vitro NMR data were acquired by using a 0.4 mM uniformly 15 Nenriched sample in 10 mM sodium acetate, pH 5.0͞10 mM sodium chloride͞0.02% sodium azide͞10.0% (vol͞vol) D 2 O at 25°C. For the crowding experiments, glucose, BSA, or ovalbumin were incorporated into the sample. The sample used for live-cell NMR spectroscopy was obtained by overexpressing 15 N-labeled FlgM in BL21 Gold Escherichia coli bacteria and by preparing as described (9). Two-dimensional gradient-enhanced ResultsThe HSQC spectrum of 15 N-enriched FlgM in dilute solution is contrasted with the spectrum in living E. coli cells in Fig. 1. In-cell NMR experiments are possible because FlgM is overexpressed upon induction (Ϸ100 mg of FlgM can be purified from 1 liter of saturated culture). The overexpression allows the FlgM spectrum to be observed on top of signals arising from other 15 N-enriched proteins in the cell, which contribute a uniform background (9). As shown in Fig. 1, about half These data suggest that the C-terminal portion of FlgM is structured in cells, but the N-terminal portion remains unstructured. We also performed important controls. ...

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Most theories about macromolecular crowding focus on two ideas: the macromolecular nature of the crowder and entropy. For proteins, the volume excluded by the crowder favors compact native states over expanded denatured states, enhancing protein stability by decreasing the entropy of unfolding. We tested these ideas with the widely used crowding agent Ficoll-70 and its monomer, sucrose. Contrary to expectations, Ficoll and sucrose have approximately the same stabilizing effect on chymotrypsin inhibitor 2. Furthermore, the stabilization is driven by enthalpy, not entropy. These results point to the need for carefully controlled studies and more sophisticated theories for understanding crowding effects.

In-cell NMR 1-3 provides information about how the crowded environment in cells, where the concentration of macromolecules reaches hundreds of grams per liter, 4 affects protein structure and dynamics. Several successes, including target protein overexpression in Escherichia coli 1,5-9 and injection of isotope-enriched protein into Xenopus laevis oocytes, 10,11 have been reported, but in-cell NMR remains in its infancy, and several potential problems need to be addressed. One problem is protein leakage from the cell during the experiment. 12-14 When this occurs, sharp signals from the protein molecules in the less viscous media mask the broader signals from the protein molecules in the more viscous cytosol. Here we examine two proteins. The intrinsically disordered protein, α-synuclein (αSN, ∼14 kDa), does not leak and is observed by in-cell NMR. The globular protein, chymotrypsin inhibitor 2 (CI2, ∼7 kDa), 15 leaks, and the remaining intracellular CI2 is not detectable. We show that the difference in detectability between αSN and CI2 is consistent with a differential dynamical response to macromolecular crowding. Figure 1A shows the 15 N-1 H HSQC spectrum of an in-cell NMR experiment on αSN. The spectrum is consistent with that from previous studies. 9,16 Figure 1B shows the spectrum from the supernatant collected immediately after sample preparation. Only metabolite signals 17 are observed. Figure 1C shows the spectrum from the supernatant recovered after the in-cell NMR experiment. Again, only metabolites are observed. The data demonstrate that the αSN spectrum in panel A comes from αSN in the cell. We have obtained similar results with the intrinsically disordered protein FlgM. 8 We performed the same experiments with CI2 expressing cells. In contrast to αSN, all three spectra are nearly identical ( Figure 1E-G) (and typical of a CI2 spectrum 18 in dilute solution). These data suggest that CI2 leaks from the cells. SDS-PAGE confirms that ∼20% of the CI2 is lost from cells. Figure 1D), proving that encapsulated cells can provide useful in-cell spectra. NIH Public AccessWe repeated the experiment with CI2-expressing cells. No CI2 signal was observed even though we increased the sensitivity by accumulating the data for a longer time compared to the other samples ( Figure 1H). However, a typical CI2 spectrum was recovered after dissolving the encapsulates with EDTA (data not shown). These observations suggest that the signal from the intracellular CI2, which we know is present in detectable amounts, is too broad to observe. We reasoned that the broadening arises from an alteration in the dynamics of CI2, either from binding a larger species in cells or from the higher viscosity of E. coli cytoplasm, which can be 10-11 times that of water. 21,22Why would the intrinsically disordered proteins αSN and FlgM react differently compared with a globular protein CI2 to the increased viscosity in cells such that we detect αSN and FlgM, but not CI2? The ability to detect a protein by high-resolution NMR depends on its dy...