Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression and transmission of mitochondrial DNA (mtDNA). However, mechanisms for the post-translational regulation of TFAM are poorly understood. Here, we show that TFAM is lysine acetylated within its high-mobility-group box 1, a domain that can also be serine phosphorylated. Using bulk and single-molecule methods, we demonstrate that site-specific phosphoserine and acetyl-lysine mimics of human TFAM regulate its interaction with non-specific DNA through distinct kinetic pathways. We show that higher protein concentrations of both TFAM mimics are required to compact DNA to a similar extent as the wild-type. Compaction is thought to be crucial for regulating mtDNA segregation and expression. Moreover, we reveal that the reduced DNA binding affinity of the acetyl-lysine mimic arises from a lower on-rate, whereas the phosphoserine mimic displays both a decreased on-rate and an increased off-rate. Strikingly, the increased off-rate of the phosphoserine mimic is coupled to a significantly faster diffusion of TFAM on DNA. These findings indicate that acetylation and phosphorylation of TFAM can fine-tune TFAM–DNA binding affinity, to permit the discrete regulation of mtDNA dynamics. Furthermore, our results suggest that phosphorylation could additionally regulate transcription by altering the ability of TFAM to locate promoter sites.

Cytochrome f (Cyt f) and plastocyanin (Pc) form a highly transient complex as part of the photosynthetic redox chain. The complex from Nostoc sp. PCC 7119 was studied by NMR relaxation spectroscopy with the aim of determining the orientation of Pc relative to Cyt f. Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc. The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.

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In this paper, we address the interpretation of molecular properties of selected singly and doubly spin-labeled peptides from continuous-wave electron spin resonance (cw-ESR) spectroscopy. This study is performed by means of an integrated computational approach that merges a stochastic treatment of long-term dynamics to ad hoc methodologies for the calculation of structural properties. In particular, our method is based on (i) the determination of geometric and local magnetic parameters of the peptides by quantum mechanical density functional calculations by taking into account solvent contribution; (ii) the hydrodynamic evaluation of dissipative properties; and (iii) molecular dynamics including equilibrium distribution of molecular conformations. The system is then described by a stochastic Liouville equation in which the spin Hamiltonian for the two electron spins, interacting with each other and coupled to two (14)N nuclear spins, is coupled to the diffusive operator describing the time evolution of slow coordinates. cw-ESR spectra are simulated for selected peptides built from the non-natural α-aminoacids α-aminoisobutyric acid (Aib) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, we study the -Aib-TOAC-Aib- singly labeled tripeptide and the -Aib-TOAC-(Aib)(7)- singly labeled and -Aib-TOAC-(Aib)(5)-TOAC-Aib- doubly labeled nonapeptides. We show that good agreement is obtained with minimal resorting to fitting procedures, proving that the combination of sensitive ESR spectroscopy and sophisticated modeling is a powerful approach to the investigation of both molecular dynamics and 3D-structural properties.

The amyloid β (A β) peptide is important in the context of Alzheimer’s disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the hydrophobic part of A β suggests a possible membrane activity of the peptide. We employed spin-label EPR to investigate the aggregation process of A β1–40 in the presence of the sodium dodecyl sulfate (SDS) detergent as a membrane-mimicking agent. In this work, the effect of SDS on A β is studied using two positions of spin label, the N-terminus and position 26. By comparing the two label positions, the effect of local mobility of the spin label is eliminated, revealing A β aggregation in the SDS concentration regime below the critical micelle concentration (CMC). We demonstrate that, at low SDS concentrations, the N-terminus of A β participates in the solubilization, most likely by being located at the particle–water interface. At higher SDS concentrations, an SDS-solubilized state that is a precursor to the one A β/micelle state above the CMC of SDS prevails. We propose that A β is membrane active and that aggregates include SDS. This study reveals the unique potential of EPR in studying A β aggregation in the presence of detergent.