Low Frequency L-Band EPR of Cu2+ in Prions

William E. Antholine, Associate Professor of Biophysics

This study focuses on obtaining improved resolution L-band EPR using data for the type-2 Cu2+ binding site in the octarepeat domain of the prion protein (PrP). PrP is the causative agent in Mad Cow disease, chronic wasting disease, and the human afflictions kuru and Creutzfeldt-Jakob disease. It is this type of problem for which EPR methods have been developed at the National Biomedical EPR Center.

Figure 1 shows four simulated spectra using EPR parameters for Cu2+ bound to the octarepeat region of PrP. The superhyperfine structure is not resolved at 9.3 GHz (not shown) and barely resolved at 4.5 GHz, resolved at 3.4 GHz, and better resolved at 1.2 GHz (Fig. 1). It is obvious from simulations that L-band can be a better microwave frequency than S-band for resolution of the superhyperfine structure in the g-parallel region.

Simulated L-band spectra

Two L-band spectra as shown in Figure 1 for Cu2+ added to a fragment that mimics a fragment in a PrP, for which the lines in both the g-parallel and g-perpendicular region were well resolved at S-band (spectrum not shown). Here, 36 scans were averaged. The superhyperfine structure is well resolved in both the g-parallel and g-perpendicular regions, but again baseline signals are similar to the signals in the gll region, so it is impossible to interpret the data in the gll region. It appears that L-band would be the frequency of choice if the SNR could be improved by at least a factor of three, or better yet, a factor of five.

In collaboration with the resonator development team, a new L-band resonator has been designed. Detailed simulations show that it will require more samples but will yield the required sensitivity at L-band. Specifically, this sample volume will increase from 0.07 ml, as used to produce Figure 2, to 1 ml. The sensitivity will increase by 6.7 times. The sensitivity at L-band relative to that presently available at S-band will increase as shown by HFSS calculations by 1.14 times. A problem was encountered in the design of the coupling structure at L-band, which was an inductive coupling, and, therefore, a capacitive coupler was designed using HFSS and demonstrated in a bench setup using a network analyzer. Construction of a new resonator has been initiated.

Characterization of the Iron-Sulfur Centers of the Bidirectional Hydrogenase 'CpI' from Clostridium pasteurianum

Brian Bennett, Associate Professor of Biophysics

The aim of this project is to deconvolute the EPR signals from the multiple [FeS] clusters in CpI, assign spectroscopic signatures to the crystallographically characterized clusters, and to elucidate the roles of the clusters in the electron transfer process. The signal from partially reduced CpI has been simulated, but the parameters remain tentative, as the species are overlapping (Figure 1).

X-band CW EPR spectra  CW EPR spectra


CW examination of a new sample (Figure 2) confirmed that CW saturation cannot effectively deconvolute the signals. Simulation at X-band (Figure 3, left) shows that only four of the nine turning points are sufficiently resolved for CW saturation analysis, and none of the

 features from the putative species C (Figure 1) are resolved. Simulation at Q-band (Figure 3, right) provides additional resolution of two of the turning points of species C.

simulated spectra

Progress has been made with the application of rapid quench EPR at Q-band. The rapid quench apparatus has been assembled and calibrated against stopped flow with MbN3 (Figure 4), and a new 35 GHz resonator has been constructed to accept 3 mm tubes. The multiquantum X-band spectrometer is being recommissioned to carry out preliminary MQ experiments at liquid helium temperatures.

calibration curves of the rapid quench apparatus



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