Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.7.1.4 (nitrite reductase)
 1,847 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Single-molecule fluorescence measurements allow researchers to study asynchronous dynamics and expose molecule-to-molecule structural and behavioral diversity, which contributes to the understanding of biological macromolecules. To provide measurements that are most consistent with the native environment of biomolecules, researchers would like to conduct these measurements in the solution phase if possible. However, diffusion typically limits the observation time to approximately 1 ms in many solution-phase single-molecule assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the observed heterogeneity. Combining the technical capabilities of high-sensitivity single-molecule fluorescence microscopy, real-time feedback control and electrokinetic flow in a microfluidic chamber, we have developed a device called the anti-Brownian electrokinetic (ABEL) trap to significantly prolong the observation time of single biomolecules in solution. We have applied the ABEL trap method to explore the photodynamics and enzymatic properties of a variety of biomolecules in aqueous solution and present four examples: the photosynthetic antenna allophycocyanin, the chaperonin enzyme TRiC, a G protein-coupled receptor protein, and the blue nitrite reductase redox enzyme. These examples illustrate the breadth and depth of information which we can extract in studies of single biomolecules with the ABEL trap. When confined in the ABEL trap, the photosynthetic antenna protein allophycocyanin exhibits rich dynamics both in its emission brightness and its excited state lifetime. As each molecule discontinuously converts from one emission/lifetime level to another in a primarily correlated way, it undergoes a series of state changes. We studied the ATP binding stoichiometry of the multi-subunit chaperonin enzyme TRiC in the ABEL trap by counting the number of hydrolyzed Cy3-ATP using stepwise photobleaching. Unlike ensemble measurements, the observed ATP number distributions depart from the standard cooperativity models. Single copies of detergent-stabilized G protein-coupled receptor proteins labeled with a reporter fluorophore also show discontinuous changes in emission brightness and lifetime, but the various states visited by the single molecules are broadly distributed. As an agonist binds, the distributions shift slightly toward a more rigid conformation of the protein. By recording the emission of a reporter fluorophore which is quenched by reduction of a nearby type I Cu center, we probed the enzymatic cycle of the redox enzyme nitrate reductase. We determined the rate constants of a model of the underlying kinetics through an analysis of the dwell times of the high/low intensity levels of the fluorophore versus nitrite concentration.
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PMID:Probing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap. 2261 16

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.
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PMID:The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway. 3072 50