Contact

Shoeman, Robert L.
Robert L. Shoeman
Phone: +49 6221 486-577
Room: R.220

Links

Swiss Light Source - SLS

The Swiss Light Source (SLS) at the Paul Scherrer Institut is a third-generation synchrotron light source. With an energy of 2.4 GeV, it provides photon beams of high brightness for research in materials science, biology and chemistry. [more]

Analytical Protein Biochemistry

Header image 1313669867

Analytical Protein Biochemistry: Robert Shoeman

The performance of instrumentation for the precise biochemical characterization of proteins has increased dramitically in recent years, primarily as a result of improvements in computer hardware. This is particularly obvious in the field of mass spectrometry, where newer instruments show increases in resolution and sensitivty of more than 1000-fold over instruments dating from the year 2000, and in robotics, where sophisticated as well as dedicated instruments have become affordable and usable, even for small groups of users.

4DX XSpectra

<p>The XSPECTRA microspectrophotometer installed at the protein crystallography X10SA beamline at the swiss light source, Villigen, Switzerland. The optical axis and the synchrotron beam axis intersect within a 20 µm spot on which the crystal is centered.</p> Zoom Image

The XSPECTRA microspectrophotometer installed at the protein crystallography X10SA beamline at the swiss light source, Villigen, Switzerland. The optical axis and the synchrotron beam axis intersect within a 20 µm spot on which the crystal is centered.

[less]

In other experiments (in collaboration with Ilme Schlichting and Jochen Reinstein Group), we have combined micro optical spectroscopy with X-ray diffraction analysis and mass spectroscopy. Our department's microspectrophotometer is a further development (originally by Georg Holtermann, Max Planck Institute of Molecular Physiology Dortmund) of the commercial 4DX XSPECTRA system and can be used in a stand-alone mode or at the synchrotron beamline (Figure 4).

This makes possible a variety of sequential measurements, two of which are shown in Figure 5. The UV-VIS spectrum of a single protein crystal of a blue light photoreceptor, the BLUF protein BlrB, was measured at 100 K, confirming that the dark adapted ground state of the receptor had been crystallized (Figure 5, left side); thereafter, the crystal was transferred to a Maldi target, washed and analyzed. Unexpectedly, 2 peaks are seen with a difference in m/z values of 785 Da. This mass difference is that expected for a FAD cofactor. Further experiments have shown that the acidic matrix solution effectively extracts much of the bound FAD (it can be found in wash solutions). Irrespective of this experimental nuance, the result presented in Figure 5 clearly shows that FAD (m/z 785) and not FMN (m/z 458) is the cofactor bound to the protein in the crystal, which is important as there is no electron density for the terminal AMP part. This observation confirms the result of conventional HPLC assays that have been performed on this and other similar proteins, but which cannot be performed on small, single crystals.

A crystal of a BLUF domain protein in various positions on the  microspectrophotometer and its UV-VIS spectrum (left). Maldi TOF  analysis of the same crystal (right) demonstrates that this protein  contains bound FAD and not FMN. Zoom Image
A crystal of a BLUF domain protein in various positions on the microspectrophotometer and its UV-VIS spectrum (left). Maldi TOF analysis of the same crystal (right) demonstrates that this protein contains bound FAD and not FMN. [less]

A crystal of a BLUF domain protein in various positions on the microspectrophotometer and its UV-VIS spectrum (left). Maldi TOF analysis of the same crystal (right) demonstrates that this protein contains bound FAD and not FMN.

 
loading content
Go to Editor View