Graded Structural Polymorphism in a Bacterial Thermosensor Protein

Abstract

Thermosensing is critical for the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family.

Switching Protein Conformational Substates by Protonation and Mutation

“…The protocols for protein purification and the majority of the spectroscopic experiments can be found in references 18 and 19. The buffers used in the pH-dependent study were glycine-HCl (pH 2–3), sodium acetate (pH 3.5–5.5), and sodium phosphate (pH 6–8).…”

“…One such example in the enterobacteriaceae is the four-helix Hha-family of proteins that regulates the availability of the transcription factor H-NS 11–15. Mutational studies16,17 and spectroscopic experiments provide strong evidence that Cnu (YdgT), a member of the Hha-family, exhibits graded structural polymorphism with temperature18 that could be critical for the expression of virulence genes in enterobacteriaceae by modulating the binding affinity with H-NS 19…”

Thiophene-S,S-dioxidized diarylethenes for light-starting irreversible thermosensors that can detect a rise in heat at low temperature

“…The crude product was purified by column chromatography on silica gel (n-hexane/ethyl acetate = 85:15) and by HPLC (n-hexane/ethyl acetate = 95:5) to give 34 mg of 1b(O) in 24% yield as a pale yellow crystal. 1…”

Tuning the Continuum of Structural States in the Native Ensemble of a Regulatory Protein

“…11 Global spectroscopic, site-specific NMR experiments, hydrodynamic measurements, theoretical modeling, and simulations indicate that the Cnu native ensemble is best described by an array of conformational states that are in dynamic equilibrium with one another in a single broad native well. If this is indeed the case, then solvent modulations with chemical denaturants together with thermal perturbations should result in nontrivial effects on the folding landscape.…”

“…In this regard, it was recently identified that the four-helix bundle protein Cnu ( Figure 1 a), a single gene product, displays thermosensor-like properties that are critical for efficient pathogenic response in enterobacteriaceae that commonly infect human gastrointestinal tracts. 11 Global spectroscopic, site-specific NMR experiments, hydrodynamic measurements, theoretical modeling, and simulations indicate that the Cnu native ensemble is best described by an array of conformational states that are in dynamic equilibrium with one another in a single broad native well. If this is indeed the case, then solvent modulations with chemical denaturants together with thermal perturbations should result in nontrivial effects on the folding landscape.…”