We are interested in structural features of membrane-associated or membrane-integrated biomolecules in general. High-resolution NMR is primarily used for our structural studies, but we complement this work with other biophysical techniques. In particular we have been interested in the process of recognition of hormones by their receptors (GPCRs). We are now also extending our work to study the receptors themselves. Recently we also started investigating a particular class of soluble proteins called repeat proteins, as scaffolds to design peptide binders.
The molecules (usually peptides or proteins) are produced recombinantly to facilitate isotope labeling, but shorter peptides are also synthesized by solid-phase peptide synthesis.
Structural Characterization of Fragments from G-protein Coupled Receptors (GPCR)
In an attempt to structurally characterize
G-protein coupled receptors we are expressing or synthesizing large fragments from
the Neuropeptide Y (the so-called Y) receptors. The fragments are produced
recombinantly as fusions to insoluble proteins or directly. The fragments are
solubilized in phospholipids micelles and structurally characterized by
high-resolution NMR spectroscopy. Presently we are developing methods to transfer assignments from these fragments onto the entire receptor. This work is initiated to improve
our understanding of the structure of these pharmaceutically highly important
receptors, as well as to shed light on the events that take place
during the assembly of the 7-TM helix bundle during folding.
One system that was recently investigated comprises constructs derived from the Y4 receptor. On another project we are determining structures of fragments from the yeast Ste2p GPCR in collaboration with Fred Naider from the City University of New York and Jeff Becker from Tennessee. We are now progressing from large fragments comprising three or more TM helices, e.g. a TM1-TM2-TM3 fragments, to the entire receptor.
Design of GPCR Mimetics (Epitope Grafting)
The aim of this project is to graft binding epitopes of the Y receptors, which represent receptors of the pharmacologically very important class of hormones of the neuropeptide Y family, onto a rigid scaffold. Typically, such scaffolds are built upon units of rigid secondary structure, and loops with intrinsic structural plasticity are targeted for mutagenesis. We have chosen beta-barrel proteins as scaffolds. So far, both soluble beta-barrel proteins from the class of the so-called lipocalins as well as beta-barrel proteins from the outer membrane of E. coli were tested. Grafting of complete loops could be successfully achieved in case of the membrane proteins. Constructs derived from the E.coli outer membrane protein OmpA successfully bind the cognate ligand NPY.
Rational design of Armadillo Repeat Proteins as a modular recognition system for the sequence-specific binding of peptides using NMR spectroscopy
The specific recognition of peptide sequences by antibodies
plays an important role in cell biology and diagnostic applications but the
production of antibodies is time-consuming. In this project in collaboration
with the groups of Andreas Plückthun, Markus Grütter and Amedeo Caflisch we aim
to develop Armadillo repeat proteins to bind extended peptides in a modular
way. Therein, each repeat represents one module binding a 'two amino
acid'-fragment of a peptide. Due to the complexity of the problem, we will
follow an interdisciplinary approach combining experiments and computation.
NMR is used to access i) the structural stability of the developed proteins, ii) to test whether peptides bind to these proteins and iii) to characterize the binding mode of the peptides. This information will be directly used for the design process of the pr
Solution Structures of Metallothioneins
We have a long-lasting interest in the structures of metallothioneins, a class of biologically relevant polypeptides comprising a very high content of Cys residues. These residues coordinate to various metal ions such as Zn,Cd,Hg,Cu. We use NMR to determine the solution strucutres of these proteins. We are particularly interested in developing methodology to better characterize the mode of metal coordination and to understand the relationship between amino acid sequence and the nature of the metal clusters.