Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli

Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation.     

Coarse-grained dynamics of the receiver domain of NtrC: fluctuations, correlations and implications for allosteric cooperativity

Receiver domains are key molecular switches in bacterial signaling. Structural studies have shown that the receiver domain of the nitrogen regulatory protein C (NtrC) exists in a conformational equilibrium encompassing both inactive and active states, with phosphorylation of Asp54 allosterically shifting the equilibrium towards the active state. To analyze dynamical fluctuations and correlations in NtrC as it undergoes activation, we have applied a coarse-grained dynamics algorithm using elastic network models. Normal mode analysis reveals possible dynamical pathways for the transition of NtrC from the inactive state to the active state. The diagonalized correlation between the inactive and the active (phosphorylated) state shows that most correlated motions occur around the active site of Asp54 and in the region Thr82 to Tyr101. This indicates a coupled correlation of dynamics in the "Thr82-Tyr101" motion. With phosphorylation inducing significant flexibility changes around the active site and alpha3 and alpha4 helices, we find that this activation makes the active-site region and the loops of alpha3/beta4 and alpha4/beta5 more stable. This means that phosphorylation entropically favors the receiver domain in its active state, and the induced conformational changes occur in an allosteric manner. Analyses of the local flexibility and long-range correlated motion also suggest a dynamics criterion for determining the allosteric cooperativity of NtrC, and may be applicable to other proteins.     

Self‐guided Langevin dynamics study of regulatory interactions in NtrC

Multiple self‐guided Langevin dynamics (SGLD) simulations were performed to examine structural and dynamical properties of the receiver domain of nitrogen regulatory protein C (NtrC^r). SGLD and MD simulations of the phosphorylated active form structure suggest a mostly stable but broad structural ensemble of this protein. The finite difference Poisson–Boltzmann calculations of the pK_a values of the active site residues suggest an increase in the pK_a of His‐84 on phosphorylation of Asp‐54. In SGLD simulations of the phosphorylated active form with charged His‐84, the average position of the regulatory helix α4 is found closer to the starting structure than in simulations with the neutral His‐84. To model the transition pathway, the phosphate group was removed from the simulations. After 7 ns of simulations, the regulatory helix α4 was found approximately halfway between positions in the NMR structures of the active and inactive forms. Removal of the phosphate group stimulated loss of helix α4, suggesting that the pathway of conformational transition may involve partial unfolding mechanism. The study illustrates the potential utility of the SGLD method in studies of the coupling between ligand binding and conformational transitions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Positive and Negative Control of Enhancer-Promoter Interactions by Other DNA Loops Generates Specificity and Tunability

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Structural and functional studies of the response regulator HupR

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.     

Topological Frustration in βα-Repeat Proteins: Sequence Diversity Modulates the Conserved Folding Mechanisms of α/β/α Sandwich Proteins

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of nonlocal interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα-repeat proteins, which are dominated by local interactions, we performed a combined experimental and simulation analysis on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured submillisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine, and valine (ILV) side chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3, and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely folded unproductive intermediates are a common property of the βα-repeat motif.