Department of Molecular Physiology

Our interests

Membrane proteins
Astacin - proteases (under construction)

The biological function of cell membranes reflects the set of membrane proteins present. Most membranes contain between 10 and 50 different major protein types, with molecular weights ranging from as little as 10000 to more than 250000 Da.

Membrane proteins contain about the same proportion of hydrophobic amino acids as the soluble proteins of the internal cytoplasm – slightly less than 50%. However the distribution of hydrophobic and hydrophilic amino acids is quite different. In most soluble proteins hydrophobic residues tend to be scattered among hydrophilic amino acids without clustering. In contrast, in membrane proteins the hydrophobic blocks of membrane proteins form segments, usually organized into an alpha helix, that are long enough to span the thickness of the membrane. These membrane-spanning segments, numbering 1 to 20 or more in different membrane proteins, form anchors that hold the proteins in stable alignment in the lipid bilayer. The transmembrane segments typically extend back and forth across the membrane, each connected to the next by a short stretch of hydrophilic amino acids that forms a loop at the membrane surface.
Some membrane proteins contain transmembrane segments in which one to three hydrophobic amino acids alternate in a repeating pattern with similar numbers of hydrophilic residues. When wound into an alpha helix, this arrangement places the hydrophobic residues on one side of the helix and the hydrophilic ones on the other side. Such alpha helices therefore have a polar and a nonpolar face. These helices occur only in membrane proteins with multiple transmembrane segments. The segments in these proteins align such that the hydrophilic sides cluster around the central axis of the protein. This arrangement creates a polar channel extending through the protein from one membrane surface to the other. Such arrangements are believed to be typical of transport proteins, which conduct charged and polar molecules across the membrane via the polar channel.
Proteins with covalently attached carbohydrate groups occur in many types of cellular membranes. The carbohydrate groups, which include essentially the same monosaccharides as those of glycolipids, link into straight or branched chains containing from 2 to 60 residues. Glycoproteins are most abundant in plasma membranes, where their carbohydrate groups occur almost exclusively on the outer membrane surface. Along with the carbohydrate groups of glycolipids, they give the cell surface what is often described as a “sugar coating” or glycocalyx. Relatively small amounts of glycoproteins also occur in internal membranes such as those of the endoplasmic reticulum (ER), Golgi complex, and nuclear envelope.
The fluid mosaic model defined two major classes of membrane proteins – integral and peripheral – according to their mode of association with membrane bilayers. Integral proteins are deeply embedded in the bilayer and held in place by nonpolar interactions with membrane lipids. Suspension in the bilayer depends on the amphiphilic properties of membrane proteins. At polar membrane surfaces, protein molecules fold to expose only hydrophilic amino acid side chains. Nonpolar side chains are exposed on the proteins surfaces facing the hydrophobic membrane interior, held in this position by their association with the nonpolar hydrocarbon chains of membrane lipids.
Integral proteins remain in stable suspension in the bilayer because, as with membrane lipids, any change in orientation would expose their hydrophobic regions to the aqueous surroundings. Because of their intimate association with the nonpolar membrane interior, integral membrane proteins can be removed from membranes only by agents such as detergents or nonpolar solvents, that disperse the bilayer. Within these limitations, integral proteins are potentially free to displace phospholipid molecules and move laterally through the fluid bilayer.
Peripheral proteins are hydrophilic molecules that bind noncovalently to polar membrane surfaces. Because of their hydrophilic nature and polar associations, peripheral membrane proteins can be removed by relatively mild treatments that do not disrupt the bilayer, such as adjustments of the salt concentration or pH of the medium.
Our work is focussed on the isolation and purification of proteins localized in the ciliary membranes of olfactory sensory neurons. To this end we use 1D gelelectrophoresis, 2D-gelelectrophoresis and affinity chromatography techniques.