Conformations and Exchange Dynamics of FlgM, an Intrinsically Disordered Protein, in Dilute and Crowded Conditions Studied by NMR Spectroscopy

Abstract

The majority of biochemical and biophysical studies are performed using dilute solutions of macromolecules. In contrast, the cellular environment contains a high total concentration (up to 400 g/L) of biomacromolecules. Concentrated bystander molecules (i.e. crowders) can exert important effects on the dynamics and conformational ensembles of proteins, especially intrinsically disordered proteins (IDPs). In particular, FlgM, a regulator of the ordered synthesis of flagellar proteins, is an IDP with transient helices in the C-terminal region and becomes structured upon binding its sigma factor target. Structure formation can be induced by high concentrations of glucose and bovine serum albumin. To investigate the conformations and exchange dynamics of FlgM in dilute and crowded conditions, we carried out backbone 15N NMR relaxation and CPMG relaxation dispersion experiments. In a dilute condition, elevated transverse relaxation rates (R2) in the C-terminal region are consistent with transient secondary structure. CPMG relaxation dispersion data indicate conformational exchange throughout the protein sequence, possibly between extended conformations with few tertiary contacts and more compact conformations with some tertiary contacts. The addition of 100 g/L dextran resulted in elevation of R2 throughout the protein, suggesting an increase in helical content in both the C-terminal and N-terminal regions. Moreover, dextran led to an increase in the amplitude of dispersion, suggesting an increase in the population of compact conformations. The NMR studies lay the foundation for quantitative characterization of an IDP in dilute and crowded conditions.