FlgM gains structure in living cells

Abstract

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. ...

The C-terminal Domain of the Measles Virus Nucleoprotein Is Intrinsically Disordered and Folds upon Binding to the C-terminal Moiety of the Phosphoprotein

“…A pioneering study has recently demonstrated that an intrinsically disordered protein region is in fact structured in living cells (73). FlgM, a 97-residue protein from Salmonella typhimurium, which regulates flagellar synthesis by binding the transcription factor 28 , is fully unstructured in vitro (74).…”

Soft interactions and crowding

“…Interestingly, HSQC spectra of disordered proteins such as FlgM and α-synuclein are easier to observe in cells (Dedmon et al 2002;). This increased detectability probably arises because disordered proteins possess much more internal motion than do globular proteins, and this internal motion is less affected by attractive interactions with other cellular macromolecules.…”

Optimizing Long Intrinsic Disorder Predictors With Protein Evolutionary Information

“…These observations support the existence of protein disorder inside the cell. The existence of intracellular protein disorder recently received additional support when FlgM, previously shown to be an intrinsically disordered protein 14,15 , was found by in vivo NMR experiments to gain some structure for only part of the molecule when in the cell, with about half of the protein remaining in the intrinsically disordered form 16 .…”