Structural characterization of intact heterogeneous protein assemblies by mass spectrometry

Summary

The overall goal of this project is to develop tools to monitor structure, dynamics, stability and interactions of heterogeneous multi-protein assemblies primarily using mass spectrometry. Two RNA processing complexes will be the target assemblies.
In the post-genomic era it is becoming increasingly evident that multi-protein complexes provide an important level of cell organization and regulation of all biological processes. The structural analysis of these heterogeneous assemblies consisting of 2 up to more than 50 different constituents is more complicated than the analysis of individual proteins. Typical problems that arise upon studying these complexes using current methods of analysis are their low abundance and their heterogeneity. An attractive novel method to study (heterogeneous) protein assemblies is by so-called ?native? electrospray ionization mass spectrometry. In recent years mass spectrometry has grown into an increasingly important technique in protein structural biology. Soft ionization methods can generate intact gas-phase ions from solutions in which proteins and protein assemblies are in their native state, allowing investigation of their structure, dynamics and non-covalent interactions. Appealing advantages of such a mass spectrometry approach are the low amount of material required and the possibility to analyze each constituent in a mixture as long as there is a difference in mass.
In this project we will structurally and functionally characterize the RNA processing assemblies exosome and degradosome from yeast by native mass spectrometry. We aim to develop useful tools for the study of a broad range of other heterogeneous protein assemblies. We will also study interactions between individual proteins and between proteins and substrates (analogues) of the mentioned protein machineries by mass spectrometry. This will give us even more detailed information on how these proteins function within the context of a macromolecular protein complex.

Output

Scientific article

  • P. Gerbaux, S. Gato, C. Kleanthous, R.H.H. van den Heuvel, A.J.R. Heck, C. Versluis(2005): Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry Nucleic Acids Research pp. e96
  • A.J.R. Heck, W.J.H. van Berkel, M.W. Fraaije, N.M. Kamerbeek, D.B. Janssen, N. Tahallah, R.H.H. van den Heuvel(2005): Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase The Journal of Biological Chemistry pp. 32115 - 32221
  • A.J.R. Heck, B. van Breukelen, A. Barendregt, R.H.H. van den Heuvel(2006): Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra Rapid Communications in Mass Spectrometry
  • S.A. Synowsky, S. Mohammed, A.J.R. Heck, R.H.H. van den Heuvel(2006): Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex Molecular and Cellular Proteomics
  • S.M. van der Vies, P.J. Bakkes, C.V. Robinson, D.A. Simmons, E. van Duijn, R.H.H. van den Heuvel, R.M. Heeren, A.J.R. Heck, H. van Heerikhuizen(2006): Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring Journal of the American Chemical Society
  • E. van Duijn, J. Hoyes, H. Mazon, R.H.H. van den Heuvel, D. Langridge, S.J. Brouns, K. Lorenzen, J. van der oost, C. Versluis, S.A. Synowsky, A.J.R. Heck(2006): Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry Analytical Chemistry

Details

Project number

700.54.402

Main applicant

Dr. R.H.H. van den Heuvel

Affiliated with

MSD Oss, Biotechnology Technical Operations

Team members

Dr. R.H.H. van den Heuvel

Duration

01/02/2005 to 12/11/2007