Funded PIER Projects 2014

Lipid-like peptides in membrane protein crystallization

Henning Tidow, Christian Löw

A crucial bottleneck in membrane protein structural biology is the difficulty in finding an optimal detergent that can solubilize integral membrane proteins (IMPs) and maintain their stability and function. Detergents are a poor membrane mimic and their common use in membrane protein crystallography may be one reason for the challenges in obtaining high-resolution crystal structures of many IMP families. We propose the use of lipid-like peptides as alternatives to detergents in membrane protein crystallography. Lipid-like peptides are short amphiphilic peptides containing a hydrophobic tail of non-polar amino acids and a hydrophilic head group. We plan to systematically investigate the effect of a library of lipid-like peptides on the solubilization, stability, activity and crystallizability of members of several IMP families.

Time-resolved crystallization of an enzymatic GAP-GTPase complex

Stefan Veltel, Markus Perbandt, Henry Chapman, Alke Meents

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. Time resolved X-ray crystallographic methods exist, but, due to both instrument availability and sensitive sample requirements, they have not been widely applied to macromolecular systems, especially for time resolutions below one second. There has been a resurgent interest in time-resolved structural science, fuelled by the recognition that both chemical and life scientist face many of the same challenges. Laue diffraction has been the main technique of choice to obtain time-resolved structural information. Nevertheless the pump-probe Laue method is highly demanding not only on the technical setup, but also on the quality of the crystal samples.

With this research project we will establish a proof-of-principle set-up to combine the pump-probe Laue method with the approach of serial femtosecond crystallography (SFX) on macromolecular sys-tems that can be triggered with caged compounds. Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. For the success of those experiments it is highly fundamental to establish conditions to synchronize molecules in a crystalline state in the sub-microsecond regime.

For the structural determination of the kinetics of enzymatic reactions we will focus on small GTPases and their co-enzymes. This system is of very high, general importance in cell biology with particular impact on disease processes, especially cancer and infectious diseases.

What teleost fish can tell but not mice - Effects of remodeling mechanisms on bone quality

Björn Busse, Dmitri Svergun, Manfred Rößle, Michael Amling

Bone has a hierarchical structure spanning the molecular (nanometer) to macroscopic length-scales. Each length-scale participates in generating bone’s fracture resistance, such that bone’s unique me-chanical properties generating both strength and toughness result from deformation and toughening mechanisms throughout its structure. Aging, disease, and other environmental triggers increase the risk of bone fracture primarily by altering the quality of the bone structure (i.e., altering the collagen cross-linking profile, mineralization distribution, architecture, etc.). Therefore, to make further clinical and therapeutic advances in the field of bone health, it is necessary to understand the multi-length-scale changes in bone quality that occur with disease and their corresponding impact on the mechanical integrity of the skeletal tissue. In general, very little is known about the mechanical and functional properties of teleost fish bones. However, fish bone has remarkably similar structural features as human bone as well as completely underinvestigated bone remodeling processes, which if understood could help us to improve and control bone remodeling in osteoporosis, while bypassing the complex signaling of osseous cells. We are convinced that there are undiscovered mechanisms over nanometer to millimeter length-scales that may help in the search for better bones and improved tissue quality by exploring the use of fish models for bone research.