Proteins

Proteins are masswise the most abundant group of molecules found in a cell. They act both as building blocks of the cellular structure and as executors of the main functions of the cell. Proteins have extremely complex structures, having developed over the years of evolution to perform specific tasks within the cell. They consist of a chain of amino acids, the length of which defines the size of the protein. There are a total of 20 different types of natural amino acids, each with the same backbone consisting of an amino group, a carbonyl group and a hydrogen atom, and a side- chain group different for each amino acid. The side-chains can be large or small in size, acidic or basic, polar or nonpolar, all depending on the amino acid in question.

The amino acid sequence, which defines a protein, is called the primary structure of the protein. It has an important role in both intermolecular and intramolecu- lar interactions. The intramolecular interactions define the final three-dimensional form of the protein – due to the electrostatic and van der Waals forces between each amino acid, the protein will fold over time to a conformation where it has the lowest possible free energy. The aforementioned forces along with the forming hydrogen bonds and a hydrophobic clustering force then stabilize the final form of the folded protein. The final three-dimensional structure of the protein is referred to as its ter- tiary structure.

When observing partial segments of the protein, it can be seen that groups of amino acids located near each other form specific types of structures. These struc- tures are referred to as the secondary structure of a protein. They are well-known spontaneously forming structures that are found in different proteins. The two most common secondary structures are the alpha-helix and the beta-sheet. When the back- bone of an amino acid chain rotates itself around the main axis in a certain fashion, it forms a so-called alpha-helical structure. A certain amount of rotation allows the side-chains of the amino acids in the same polypeptide chain to support each other, creating a stable molecular structure common in the majority of proteins bound in- side a lipid bilayer. Another example can be seen when a protein folds in such a way that amino acid groups run parallel to each other. Often in such situations, an exten- sive hydrogen bond network forms between two groups of laterally adjacent amino acids, creating a sheet-like structure. This type of a structure is called a beta-sheet, common in many barrel-like proteins that are capable of transporting a molecule.

Membrane proteins can be divided into two main subgroups depending on how they interact with the membrane: peripheral membrane proteins and integral mem- brane proteins. Peripheral membrane proteins are a group of proteins that have only temporary interactions with the surface of the membrane as they perform their bi- ological functions. Upon making contact with the membrane surface, the tertiary structure of a peripheral membrane protein may change as it interacts with the lipids and other membrane-bound proteins. This usually involves conformational changes around the binding region exposing the nonpolar regions of the protein with which the binding is made possible. These conformational changes depend on the biolog- ical function of the protein and may include, for instance, the opening of a ligand- binding channel, unfolding of an anchor region, or dissociation of a transported molecule. The ability to bind to a membrane typically requires the peripheral mem- brane protein to be in a specific orientation near the binding site, and a certain lipid composition or the presence of a binding protein at the target location. Many periph- eral membrane proteins are typically associated with transporting of molecules from one cell to another – they are able to bind a molecule at the surface of a membrane, and transport it to another membrane where the molecule in question is dropped off. These types of interactions also have a large significance in cellular signaling [34].

Integral membrane proteins are mainly bound to the interior of the membrane with parts of the protein protruding from the membrane surface. Transmembrane proteins are a major subgroup of integral membrane proteins. They cross the whole membrane, often multiple times, thus making contact with both the intra- and the extracellular sides of the membrane. The polypeptide chain that crosses the mem- brane is generally in an alpha-helical conformation, but many transmembrane pro- teins with a beta-barrel structure also exist. Multi-pass transmembrane proteins of- ten act as channels for ions and other substances as they are able to create an ener- getically favorable environment for these substances to traverse in their interior and vicinity when compared to a pure lipid bilayer.

The function of many transmembrane proteins also involve a change in their con- formation. Ion channel proteins among others use mechanic gating to control the flux of molecules between the membrane interior and exterior. Certain potassium channels, for instance, are activated when intracellular calcium interacts with an in- tracellular part of the channel protein, triggering a change in the conformation of

the protein[35, 36]. The gate controlling entry from the extracellular side into the channel, surrounded by many transmembrane alpha-helices, opens for a short while and potassium ions are able to enter the protein interior. After a time, the protein conformation changes again in a way that the extracellular side closes and the intra- cellular side opens, releasing the ions into the cell interior. This mechanism is vital for the function of many different cell types, especially prominent during conduc- tion of signals in nerve cells.

Another major group of transmembrane proteins are the G protein-coupled re- ceptors (GPCRs). They have a similar overall archistructure consisting of seven transmembrane alpha-helices spanning the membrane, creating a cylindrical struc- ture[37]. The main differences in their overall structures lie in their extracellular parts which are able to interact with various protein-specific ligands. These interac- tions in turn activate the protein. Instead of acting as transporters, their main func- tion lies in signal transduction. Upon binding of an extracellular signal molecule, the GPCR undergoes a conformational transformation enabling it to activate a G protein – a trimeric GTP-binding protein – which couples the GPCR to enzymes or other proteins, triggering protein-specific functions.

There are nearly a thousand different GPCR proteins, all with a specific duty in cellular life[38]. Many GPCRs are involved with the function of the senses. Opsin, for instance, is able to bind a retinal-ligand inside its transmembrane structure, thus converting itself to rhodopsin. By changing the isomerization of the retinal by ab- sorbing light, the protein changes its conformation initiating the visual transduction process[39].

GPCRs are also prevalent in the nervous system. These receptors are able to bind neurotransmitters – chemicals created at the synapses of the nervous system – which activate the protein. The GPCR then is able to control nearby ion channels, thus regulating the charge of the cell[40]. This kind of GPCR activation controls the function of the sympathetic and parasympathetic parts of the nervous system. Some GPCRs in the brain control the behavior and the mood of a person. Dopamine, for instance, is a neurotransmitter created in the synapses of the central nervous system.

Dopamine receptors are GPCRs found in the brain able to bind dopamine as an external ligand, which then triggers a typical GPCR signaling pathway resulting in control over memory, motivation and other executive functions[41, 42].

Due to their large variety and importance in life, GPCRs are major drug targets.

Currently approximately 34 % of all approved clinical drugs in the US are targeting over a hundred different GPCRs[18]while ten years earlier the number was 27 % [43]. GPCR drug discovery is the most intensively studied area in drug research with significant progress constantly made in order to identify new targets.

It is worth noting that proteins can function in more ways than their designated group indicates. For instance, recent studies have shown that many proteins of the GPCR family have secondary functions as lipid scramblases – they actively induce lipid translocation across the bilayer, thus scrambling the lipid composition of the two opposing leaflets[12, 44]. One such GPCR is rhodopsin, the protein mainly in- volved in visual transduction. The transmembrane alpha-helices provide a favorable environment for the hydrophilic lipid head groups giving them easier entry to the membrane interior. There are also indications that the peripheral myelin protein P2, a member of the fatty acid binding (FABP) family, acts as a lipid transporter as is typical for fatty acid binding proteins, but also has an important role in holding the myelin sheath – a membrane complex vital for the nervous system – together [6]. These secondary functions of both rhodopsin and P2 are further explored in the following chapters of this Thesis.