Research

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. The molecules (usually peptides or proteins) are produced recombinantly to facilitate isotope labeling, but shorter peptides are also synthesized by SPPS.

Recognition of hormones by their membrane-imbedded receptors: We have recently been working on the recognition of G-protein coupled receptors by hormones. For our studies we have characterized the neuropeptide Y (NPY)/Y-receptor system. NPY is the most abundant neurohormone in the central nervous system. For binding of NPY to its receptors we have postulated a three-step model, which includes the membrane association as an important step that precedes receptor binding of the ligand. The conducted analysis comprised the following steps: Elucidation of the structure of micelle-bound peptides Determination of the membrane-binding topology by 15N,1H correlation spectroscopy utilizing micelle-integrating spinlabels Determination of the dynamics of membrane-bound peptides by 15N relaxation By comparison with the solution structure of NPY we discovered that structural changes are introduced upon binding to the membrane. In particular, residues which are known to mediate receptor contacts become located close to the membrane, water interface in a well defined manner. We have recently been looking into possible structural determinants for receptor subtype specificity. Firstly, we have characterized the solution structure of [31Ala,32Aib]-NPY, a mutant that selectively binds to the Y-5 receptor and induces increases in food-intake. We discovered larger structural differences in the C-terminal part of the a-helix. Furthermore, we have been looking at structural differences of another Y-5 selective mutant [31Ala,32Pro]-NPY in its membrane-bound form and compared it to wild-type NPY. Presently, we investigate a number of other, subtype-specific ligands in order to verify our postulate. We have also determined the structure and dynamics of the pancreatic polypeptide (PP), a peptide that selectively targets the Y4 receptor subtype. Moreover, we compared NPY and PYY, two pharmacologically closely related peptides, and discovered that similarities in pharmacology are better related to structural features of the membrane-bound state. We have also looked into NPY/PP chimaera with interesting biological properties in order to yield a better understanding of receptor subtype selectivity. We are now looking into structures of N terminal fragments of Y receptors and test binding of these constructs to wild-type hormones in order to understand to which extent contacts with the N-terminal domain of the Y receptors may be responsible for receptor recognition and binding.

Cell-cell recognition is an event of prime biological importance in a variety of biological phenomena such as cell migration, organ formation, immune defense and microbial infection. Specificity of the interaction is mediated by various, tissue-dependent, receptors.We have initiated studies to create a functional model for cell-surface exposed carbohydrate units that can be used to investigate interactions with lectins, and which help to increase the complexity of our membrane models. We have started our studies with systems of the following architecture: A lipid anchor is extended by an flexible linker, onto which the carbohydrate portion is coupled. We have recently been been able to show that these systems are properly intergrated into our phospholipid micelles.

We have also investigated binding of Cyanovirin (CNV) to micelle-immobilized 1,2-dimannoside and 1,2 linked trimannoside and could demonstrate that the protein is properly recognized. CVN is a cyanobacterial protein that interacts through high-affinity carbohydrate-mediated interactions with the surface-envelope glycoprotein gp120 from various HIV and SIV strains thereby disrupting the gp120-CD4 interaction and blocking HIV entry. We could also demonstrate that CVN linked to micelles could be released by adding a higher affinity ligand such as the (non-lipidated) trimannoside. By adding lectin domains to proteins a system capable of reversible anchoring proteins onto membranes is created that circumvents problems with adding chemical moieties onto proteins.

Transportation of peptides across cell membranes is a common problem for pharmaceutical industry. A number of strategies have recently been developed to transfer peptides that are biologically active across membranes. In principle, two strategies have been pursued: 1) a short peptide sequence is fused to the peptide of interest. Successfully used sequences have been the TAT protein from human HIV-1 virus, the third a-helix of the Antennapedia homeodomain and the VP22 protein from HSV. In this approach the signaling sequence is covalently linked to the peptide. 2) Alternatively, a peptide carrier may be used. Such a carrier binds the peptide of interest and the resulting complex is then internalized.

We have investigated, to which part of the proteins these carriers bind and by which mechanism the cargo is carried through the membrane. We propose that changes in membrane-morphology are introduced by the peptide, which are due to i) compensation of headgroup charges through cationic residues leading to concave curvature of the membrane and ii) insertion of chemical moieties into the headgroup space resulting in convex curvature.

Organic Chemistry Institute

Research