Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver.

Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi-dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O2 consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.

Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET).

Fluorescence Resonance Energy Transfer (FRET) is a well-known methodology for detection and quantitation of structural changes of proteins in solution. FRET requires site-specific protein labeling with two fluorophores, one of which functions as an energy donor and the other one as an energy acceptor. However, the site-specific labeling of protein is often complex and difficult, particularly when inserting two fluorophores in specific sites. We have examined several protein labeling approaches with a varying degree of success. Described here is a dual labeling strategy that worked reproducibly in a number of protein targets and we believe will be applicable to a variety of proteins, which have few or no native cysteine (Cys) residues. We have successfully double-labeled DnaA protein of Bacillus anthracis, which lacks intrinsic Cys residues. A cysteine residue was inserted at the N-terminus by in vitro mutagenesis and a Cys-Cys-Phe-Gly-Cys-Cys (CCPGCC) sequence at the C-terminus by PCR. This protein was labeled site-specifically with a fluorescein derivative, FlAsH, at the CCPGCC sequence followed by Alexa568 maleimide at the N-terminus Cys residue. Structural changes of the protein with nucleotide, DNA and an inhibitor protein binding were determined by FRET analysis of the double-labeled protein. This comprehensive novel methodology for site-specific protein labeling with different fluorophores is applicable for understanding different in vitro proteomic structural studies. Here, we describe a verified technique used for FRET spectral analysis and quantitative evaluation of structural changes using fluorophore labeled DnaA protein constructs as an example.