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Switching Protein Conformational Substates by Protonation and Mutation.

ABSTRACT: Protein modules that regulate the availability and conformational status of transcription factors determine the rapidity, duration, and magnitude of cellular response to changing conditions. One such system is the single-gene product Cnu, a four-helix bundle transcription co-repressor, which acts as a molecular thermosensor regulating the expression of virulence genes in enterobacteriaceae through modulation of its native conformational ensemble. Cnu and related genes have also been implicated in pH-dependent expression of virulence genes. We hypothesize that protonation of a conserved buried histidine (H45) in Cnu promotes large electrostatic frustration, thus disturbing the H-NS, a transcription factor, binding face. Spectroscopic and calorimetric methods reveal that H45 exhibits a suppressed p Ka of ?5.1, the protonation of which switches the conformation to an alternate native ensemble in which the fourth helix is disordered. The population redistribution can also be achieved through a mutation H45V, which does not display any switching behavior at pH values greater than 4. The Wako-Saitô-Muñoz-Eaton (WSME) statistical mechanical model predicts specific differences in the conformations and fluctuations of the fourth and first helices of Cnu determining the observed pH response. We validate these predictions through fluorescence lifetime measurements of a sole tryptophan, highlighting the presence of both native and non-native interactions in the regions adjoining the binding face of Cnu. Our combined experimental-computational study thus shows that Cnu acts both as a thermo- and pH-sensor orchestrated via a subtle but quantifiable balance between the weak packing of a structural element and protonation of a buried histidine that promotes electrostatic frustration.

SUBMITTER: Narayan A

PROVIDER: S-EPMC6314467 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Switching Protein Conformational Substates by Protonation and Mutation.

Narayan Abhishek A Naganathan Athi N AN

The journal of physical chemistry. B 20180810 49

Protein modules that regulate the availability and conformational status of transcription factors determine the rapidity, duration, and magnitude of cellular response to changing conditions. One such system is the single-gene product Cnu, a four-helix bundle transcription co-repressor, which acts as a molecular thermosensor regulating the expression of virulence genes in enterobacteriaceae through modulation of its native conformational ensemble. Cnu and related genes have also been implicated i ...[more]

PMID: 30048131

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Project description:Infrared spectra of heme-bound CO in sperm whale carbonmonoxy myoglobin and two mutants (H64L and H97F) were studied in the pH range from 4.2 to 9.5. Comparison of the native protein with the mutants shows that the observed pH effects can be traced to protonations of two histidine residues, H64 and H97, near the active site. Their imidazole sidechains experience simple, uncoupled Henderson-Hasselbalch type protonations, giving rise to four different protonation states. Because two of the protonation states are linked by a pH-independent equilibrium, the overall pH dependence of the spectra is described by a linear combination of three independent components. Global analysis, based on singular value decomposition and matrix least-squares algorithms enabled us to extract the pK values of the two histidines and the three basis spectra of the protonating species. The basis spectra were decomposed into the taxonomic substates A(0), A(1), and A(3), previously introduced in a heuristic way to analyze CO stretch spectra in heme proteins at fixed pH (see for instance, Biophys. J. 71:1563-1573). Moreover, an additional, weakly populated substate, called A(x), was identified. Protonation of H97 gives rise to a blue shift of the individual infrared lines by about 2 cm(-1), so that the A substates actually appear in pairs, such as A(0) and A(0)(+). The blue shift can be explained by reduced backbonding from the heme iron to the CO. Protonation of the distal histidine, H64, leads to a change of the infrared absorption from the A(1) or A(3) substate lines to A(0). This behavior can be explained by a conformational change upon protonation that moves the imidazole sidechain of H64 away from the CO into the high-dielectric solvent environment, which avoids the energetically unfavorable situation of an uncompensated electric charge in the apolar, low-dielectric protein interior. Our results suggest that protonation reactions serve as an important mechanism to create taxonomic substates in proteins.

Dataset Information

Switching Protein Conformational Substates by Protonation and Mutation.

Publications

Switching Protein Conformational Substates by Protonation and Mutation.

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