Protein Dynamics and Monomer−Monomer Interactions in AntR Activation by Electron Paramagnetic Resonance and Double Electron−Electron Resonance

Abstract

The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

“…This is strong evidence that the peaks at 4.8 nm do not reflect dipolar interaction within the same RNA molecule, but are due to the interaction with background spins or have to be attributed to the presence of a fraction of unfolded aptamers. In addition to the analysis by Tikhonov regularization, all data were fitted with a Monte Carlo/SIMPLEX algorithm assuming a sum of Gaussian-distributed conformers contributing to the dipolar evolution data by use of the software DEFit 3.9 (Sen et al 2007). The resulting distance distributions (data not shown) resemble those obtained by Tikhonov regularization.…”

“…Interspin distance distributions were derived by fitting the dipolar evolution function using Tikhonov regularization, a commonly used method of regularization of ill-posed problems, as implemented in DEERAnalysis2006 (Jeschke et al 2006). In addition, the experimental data were fitted with a Monte Carlo/SIMPLEX algorithm assuming a sum of Gaussian-distributed interspin distances contributing to the dipolar evolution data by use of the software DEFit 3.9 (Sen et al 2007). …”

Organization of the Mitochondrial Apoptotic BAK Pore

“…Data were analyzed primarily with DeerAnalysis2006 (26) to extract the distance information. DEFit program (27) or the Global Analysis program (28) were also used to check the analysis result by the DeerAnalysis2006.…”

Electron Spin Resonance Shows Common Structural Features for Different Classes of EcoRI–DNA Complexes

“…[19][20][21] Recently, the DEER experiment has been used to probe structural rearrangements upon metal binding in the anthracis repressor, a DNA binding protein. [22] The four-pulse DEER data for each of the mutant complexes are shown in Figure 2 a. The time-domain signals were inverted to obtain the distance distribution functions, using a Tikhonov regularization method in the DEERAnalysis2006 program.…”