CW EPR spectroscopy

The trans-activation response (TAR) RNA is a structural motif at the 5Cend of the HIV RNA, which interacts with the Tat protein during HIV transcription. The binding site of the Tat protein is an internal loop in the TAR RNA (Fig. 15.10A). The TAR—Tat interaction assures an efficient transcription and thereby facilitates replication of the HIV virus (Frankel, 1992). Therefore, interference with the TAR—Tat interaction has been pursued as a strategy to combat HIV. Below is a description of how cw-EPR spectroscopy has yielded information about RNA dynamics that are correlated with the structure of the TAR RNA receptor bound to various ligands. 

It should be noted that EPR spectroscopy in fields like bioinorganic chemistry or protein research is nowadays often combined with other experimental and simulation methods. In contrast, for rather randomly structured materials such as thermoresponsive polymer systems presented here, one can obtain meaningful insight with simple CW EPR methods. Before the specific recent examples are presented, the necessary basics of CW EPR spectroscopy are explained in the next section. 

This section gives a concise introduction to CW EPR spectroscopy and only highlights the points needed for understanding the examples from polymer science in the following section. For a more detailed introduction to EPR spectroscopic techniques the reader is referred to [19, 34]. 

In this section, the EPR spectroscopic characterization of thermoresponsive polymeric systems is presented. The polymeric systems are water-swollen at lower temperatures and upon temperature increase the incorporated water is driven out and the system undergoes a reversible phase separation. Simple CW EPR spectroscopy (see above), carried out on a low-cost, easy-to-use benchtop spectrometer, is used here to reveal and characterize inhomogeneities on a scale of several nanometers during the thermal collapse. Further, neither any physical model of analysis nor chemical synthesis to introduce radicals had to be utilized. Adding amphiphilic TEMPO spin probes as guest molecules to the polymeric systems leads to self-assembly of these tracer molecules in hydrophilic and hydrophobic regions of the systems. These probes in different environments can be discerned and one... 

Almost naturally, CW EPR spectroscopy can also contribute to rmderstanding the electronic properties of polymers that are envisioned for application in the fields of polymer electronics (e.g., [98,99]) and photovoltaics (e.g., [100-102]). Unlike in the smdies highlighted before, in these cases aU materials are EPR active and paramagnetic probes do not have to be added. Going beyond the conventional use of simple CW EPR spectroscopy to study electronic defects, Van Doorslaer, Goovaerts, Groenen, and coworkers have used multifrequency (X-, Q-, and W-band) EPR techniques such as HYSCORE and pulse ENDOR (see Sect. 2.1) to elucidate the extension of polarons in films of electro-active polymers [103]. 

Each single cysteine mutant was spin labeled and analyzed by RT CW EPR spectroscopy. As an example of a nitroxide scan, spectra detected in the first (22-32) and second (86-109) segments are shown in Pig. 2b. Interestingly, being a homotetramer, each single cysteine mutant provides four spins per channel. If the distance between the four spin labels in the tetramer is on the order of 2 nm or higher, no dipolar interaction appears thus the features of the room temperature spectra purely reflect the spin label dynamics. 

Keyvrords CW EPR spectroscopy Dynamics ENDOR ESEEM Hyperfine spectroscopy In-cell EPR Nucleic acids PELDOR RNA-ligand interactions RNA-protein interactions Stmcture... 

CW EPR spectroscopy is extensively used to probe local and global dynamics of nucleic acid molecules and to correlate these data with NA structures and functional aspects. This is particularly useful to investigate and monitor NA-protein or NA-ligand interactions. In the following we are going to highlight some recent applications of CW EPR to study RNA and DNA molecules. 

RNA-protein interactions play a central role in cellular processes therefore the investigation of structure, conformational dynamics of such complexes, and their relation to biological function is of major importance. Again, CW EPR spectroscopy can help to provide detailed understanding of such processes and we will state two selected examples in the following. 

It is important to note that, despite the above remarks, the recording of the ESE-detected EPR spectra is still favored over cw-EPR spectroscopy to determine the lowest principal g value in low-spin ferric systems with high g anisotropy (supplementary material of [44]). Furfliennore, for type I systems, 2D nutation spec-... 

One obvious way to determine the zero-field splitting parameter, D, wifli cw-EPR spectroscopy, is to go to higher microwave frequencies, where die hv/D ratio becomes larger than 1. E. Reijerse, W. Hagen, and coworkers measured the cw-EPR spectra of aquometmyoglobin from 1 to 285 GHz [51]. As die microwave frequency approaches the zero-field splitting, the value of gr,eff reduces notably, giving direct information on the D value. From 130 GHz onwards, an anomalous increase of the linewidth is observed that relates directly to D strain effects. 

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