Improving Methods for X-ray Absorption Spectroscopy Studies of Metalloproteins

X-ray absorption spectroscopy (XAS) is a widely used technique within biochemistry to analyze the structure around metal ions in proteins. A spectrum consists of two parts; the X-ray Absorption Near Edge Structure (XANES) and the Extended X-ray Absorption Fine Structure (EXAFS), which both give information about the local structure around a specific element (e.g. copper), but are analyzed in different ways. The use of XAS in biochemistry is complicated by radiation damage, e.g. reduction of Cu(II) to Cu(I).
In this thesis, the preliminary work on a microfluidics flow cell for the new XAS beamline, Balder, at the MAX IV Laboratory synchrotron in Lund, Sweden is presented, and the flow capabilities are proven sufficient to avoid radiation damage with the flux expected for the beamline.
To explore the advantages of XAS in the analysis of metalloproteins, data was collected on Anabaena Variabilis plastocyanin, a protein well described in literature using X-ray diffraction giving ultrahigh resolution crystal structures. The XAS data showed clear indication of photoreduction, and the fitted models bare close resemblance to the crystal structures, thus indicating that the crystal structures published are also a result of radiation damage. The data is compared to previously collected data with little to no photoreduction, and a difference in histidine distances are found, although the data quality is lower for this dataset. In an attempt to improve the fitting procedure, a Design of Experiment was utilized to vary the fitted parameters in a statistical feasible way. While promising initial results showed good fits to the data, the time limit of this thesis did not allow for a better fit than achieved by conventional methods to be found.
XAS was also deployed to analyze the binding of Cu(II) and Zn(II) to the amyloid-β peptide (Aβ), related to Alzheimer’s Disease, and two variants of it with a mutation on the second position. For Cu(II)Aβ, the fibrillar coordination was found to depend on pH and the variant, with several models giving reasonable fits, possibly as a result of a mixture of several coordinations in the solution. For Zn(II)Aβ, the coordination was found to be the same across pH and variant, and was best fitted with a tetrahedral model of two histidines, a carboxylic group of either a glutamate or aspartate, and another oxygen or nitrogen ligand.