Biophysics of Membrane Proteins
Projects
1. Thermodynamics of intramembrane protein folding
- Figure 1: Mistic is a small bacterial membrane protein consisting of four transmembrane helices. This protein lends itself particularly well to thermodynamic investigations because it can fold autonomously in aqueous solutions, lipid membranes, and detergent micelles, thus allowing for a direct comparison among the folding processes in these environments.
Protein–protein, protein–lipid, and lipid–lipid interactions in the anisotropic environment of biological membranes control the folding, oligomerisation, and intramembrane substrate binding of membrane proteins. Highly specific contacts between transmembrane helices play a central role in these processes. Unlike in the case of soluble proteins, however, the underlying thermodynamic forces are only poorly understood on a quantitative level. Our group aims at unravelling the following questions:
- How avid and how specific are intramolecular interactions in a membrane protein? Which enthalpic and entropic factors contribute to affinity and specificity? How do these features relate to the structure of a membrane protein?
- Can we derive information about the molecular architecture of a membrane protein from interhelical binding affinities? How does this approach reflect structural differences between proteins sharing the same topology?
- Which forces drive intermolecular interactions such as oligomerisation and substrate binding in a lipid membrane? Can we pinpoint binding and catalytic sites in the membrane interior? Do different substrates of the same enzyme show different binding affinities?
We are using calorimetric and spectroscopic methods to characterise the thermodynamics of protein folding and protein–protein interactions in lipid membranes and detergent micelles. As a first step in this direction, the assembly of the simple bacterial membrane protein Mistic is assessed by systematically quantifying the interactions among four synthetic peptides corresponding to the protein’s four transmembrane domains (Figure 1). This protein lends itself particularly well to thermodynamic investigations because it can fold autonomously in aqueous solutions, lipid membranes, and detergent micelles, thus allowing for a direct comparison among the folding processes in these environments.
2. Domain formation in complex membranes
Lateral inhomogeneities in cell membranes, usually referred to as “lipid rafts”, are under intense investigation because of their involvement in numerous physiological and pathological cellular processes. Functional rafts in vivo have often been believed to be identical to detergent-resistant membrane fragments and liquid–ordered phases, but this conception is being abandoned on both experimental and theoretical grounds. Instead, proteins are being recognised as playing a decisive role in the formation of membrane domains that are too small both to be visualised by microscopic methods and to be treated as thermodynamic phases. We are particularly interested in the following issues:
- How large are protein-tethered membrane domains? Do proteins differing in size, topology, charge, or amphipathicity give rise to different kinds of domains? How is information about the aggregational state of one leaflet conveyed to the opposing leaflet?
- How do these domains relate to detergent-resistant membrane fragments? Can detergent solubilisation lead to artifactual appearance, disappearance, or alteration of domains? Which conclusions can be drawn from detergent extraction experiments?
- To which extent can peptides and proteins modulate membrane curvature? How is this reflected in the susceptibility of a mixed membrane to detergent solubilisation? Which parameters can we derive to quantify this process?
Using novel microcalorimetric techniques and confocal laser scanning microscopy, we are examining the properties of peptide- and protein-dependent lipid clusters. Poly(L-lysine) peptides of different chain lengths are useful model systems for studying the influence of proteins on lipid demixing, transbilayer coupling, and membrane curvature (Figure 2).
- Figure 2: Preliminary results with poly(L-lysine) indicate that the peptide chain length has a dramatic effect on transbilayer coupling. Whereas short peptides can sequester negatively charged phospholipids only within the accessible leaflet, adsorption of longer peptides to one side of the membrane leads to the formation of domains that span both bilayer leaflets.
