BME 220, Spring 2005
David M. Ng
Created on: 2005 April 19
This Web page is my solution for Homework 1: Making Pictures for BME 220, Spring 2005. The two proteins I selected are potassium channels and bacteriorhodopsin.
The images were created with RasMol [1] using PDB structure files from the RCSB Protein Data Bank [2].
The images in this section were created from the PDB structure file for 1BL8, "Potassium Channel (Kcsa) From Streptomyces lividans". The primary citation is by MacKinnon [3].
The 2003 Nobel Prize in Chemistry was awarded for discoveries concerning channels in cell membranes. Roderick MacKinnon was recognized for his work on ion channels. MacKinnon shared the Nobel Prize that year with Peter Agre who worked on water channels.
A channel is an integral membrane protein that provides facilitated diffusion of polar substances. Channels allow diffusion of a specific polar molecule, and they may be gated to be open or closed.
Potassium channels conduct potassium ions across the cell membrane along the concentration gradient. They are found in all domains of life, and are present in all membranes. All potassium channels are members of a single protein family. Potassium channels serve many roles including regulation of metabolism (e.g., insulin secretion) and signalling in the nervous system (e.g., the venom of the black mamba snake affects the potassium channels and can cause paralysis or death). Genetic disorders can be caused by defects in genes for potassium channels (e.g., deafness can be caused by a defect in the KCNQ4 gene).
The potassium channel is composed of four identical subunits. The figures below shows the four subunits in different colors.
The atomic radius of a potassium ion is 1.33 Angstroms; the atomic radius of a sodium ion is 0.95 Angstroms. Yet the potassium channel can select for potassium ions over sodium ions by a factor of over 1000, and at a rate close to the diffusion limit.
Ions are hydrated outside the cell. The positive ions are attracted to the potassium channel by negatively charged residues. The channel overcomes the electrostatic repulsion of the membrane, and the ions move through the channel to a water-filled cavity.
Selection of potassium ions is achieved by a selectivity filter, shown in magenta in the figures below. Each of the four subunits has a selectivity filter region. Figure 2.1 shows the "ion's view" of the potassium channel, showing how the four selectivity filter regions are arranged into a tunnel through which ions must pass.
Figure 2.1. Top view of a potassium channel (from the extracellular side of the plasma membrane). Each chain of the channel has a different color (blue, cyan, green, and yellow). The selectivity channel is shown in magenta. The glycine gating hinges are shown in orange.
Figure 2.2 shows a side view of the potassium channel, showing the length of the tunnel.
Figure 2.2. View of a potassium channel looking down at an angle (the extracellular side is at the top). Each chain of the channel has a different color (blue, cyan, green, and yellow). The selectivity channel is shown in magenta; this view shows the length of the channel. The glycine gating hinges are shown in orange.
Figure 2.3 shows negatively charged residues (that attract positively charged ions towards the channel) in blue; note the positioning of such residues near the top of the channel, particularly at the top of the selectivity filter.
Figure 2.3. Side view of potassium channel (the extracellular side is at the top). The selectivity channel is shown in magenta. Negatively charged residues are shown in blue. Note the negatively charged residues near the top of the channel, particularly at the top of the selectivity filter.
The selectivity filter works by mimicking the hydration encountered by the ion outside the cell. When the ion enters the channel, it becomes dehydrated. This would normally incur an energy cost. The selectivity filter has four weak binding sites for ions as they are passing through. At each binding site the ion is surrounded by eight oxygen atoms that provides the same electrical environment for a potassium ion as when it is hydrated, in essence simulating the hydration shell of the ion when it is outside the cell. The oxygen atoms occur in a region that is highy conserved in the potassium channel family. The positioning of the oxygen atoms simulates the hydration shell for a potassium ion, but not other ions. Figure 2.4 shows the oxygen atoms of the selectivity filter in blue, showing the binding sites for the ions.
Figure 2.4. Side view of potassium channel (the extracellular side is at the top). The selectivity channel is shown in magenta. Oxygen atoms of the selectivity filter are shown in blue. Note how the oxygen atoms are arranged in layers, providing binding sites for potassium ions to simulate hydration.
Each subunit of the potassium channel has a helix that serves as a gate that can be in the open or closed conformation. The gate helix has a glycine that serves as a hinge for the gate (glycine has only a single hydrogen as its "side chain", allowing it to pivot as a hinge). This glycine is highly conserved across the potassium channel family. The gate helices block passage of potassium ions by narrowing the channel to 3.5 Angstroms; additionally, the gate helices are lined with hydrophobic residues.
Figure 2.1 (above) shows the glycine gating hinge of each subunit in orange. The potassium channel is shown with the gate helices in the closed conformation.
Figure 2.5 shows the gate helices with the position of the glycine gating hinge shown in orange (the cyan-colored subunit shows this the most clearly). The gate helix is shown in the closed conformation.
Figure 2.5. Side view of potassium channel (the extracellular side is at the top). Each chain of the channel has a different color (blue, cyan, green, and yellow). The selectivity channel is shown in magenta. The glycine gating hinges are shown in orange; this view shows the location of the hinge on the inner helix that serves as the gate (the gate is in the closed conformation).
There are two methods of controlling the opening and closing of the gate:
The structures for gate control are not shown in the figures.
The images in this section were created from the PDB structure file for 1BRD, "Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy" from Halobacterium halobium (also known as Halobacterium salinarum). The primary citation is by Henderson, et al. [4]
Bacteriorhodopsin is a protein in some archaea that detects light using a small chromophore (light-absorbing group) named retinal that is embedded in the bacteriorhodopsin. The retinal gives the protein a purple color. Retinal changes its shape when it absorbs a photon, inducing a cis-trans isomerization. The shape change of retinal causes the bacteriorhodopsin to undergo a conformational change resulting in a proton being pumped out of the cell. Bacteriorhodopsin is a light-driven proton pump used for energy conversion, converting light to a proton gradient. The proton gradient is used to synthesize ATP. When a photon is absorbed, a proton is pumped from the cytoplasmic side of the membrane to the extracellular side. Bacteriorhodopsin is structurally similar to the G-protein-linked receptors, but since it is not coupled to a G-protein it is not actually a type of G-protein-linked receptor. Like G-protein-linked receptors, bacteriorhodopsin is a seven-spanning multipass protein that is sometimes though of as a simple model of G-protein-linked receptors.
Retinal is the aldehyde form of vitamin A, and is the same chromophore as found in rhodopsin molecules found in photoreceptor cells of the vertebrate eye.
The G-protein-linked receptors form the largest family of cell-surface receptors. They are the most numerous receptors in all eukaryotic genomes (1-5% of the total number of genes). G-protein-linked receptors transduce a variety of extracellular signals such as light, nucleotides, hormones, lipids, and proteins.
All G-protein-linked receptors share a common structure: the consist of a single polypeptide chain with seven alpha helices that pass through the plasma membrane. Bacteriorhodopsin also has this structure. Figure 3.1 shows that bacteriorhodopsin has seven helices. The figure seems to show seven separate structures, but they actually are made of a single chain (the summary information for 1BRD lists only a single chain). The C-terminal end (marked with red) is on the cytoplasmic side of the membrane.
Figure 3.1. End view of bacteriorhodopsin showing seven alpha helices characteristic of a seven-spanning multipass protein. Hydrophobic residues are colored yellow. Red marks the C-terminal end of the protein. All other residues are colored blue.
In a G-protein-linked receptor, the seven alpha helices pass through the membrane; this is also true for bacteriorhodopsin. In Figure 3.2, observe that the helices are predominantly hydrophobic (hydrophobic residues are marked with yellow), as expected for a transmembrane segment.
Figure 3.2. Side view of bacteriorhodopsin. Hydrophobic residues are colored yellow. Red marks the C-terminal end of the protein. All other residues are colored blue. This figure shows that the alpha helices are predominantly hydrophobic as expected since the seven helices pass through the membrane.
The seven alpha helices form a pocket that encloses the retinal. Figure 3.3 shows an end view of bacteriorhodopsin, showing the pocket.
Figure 3.3. End view of bacteriorhodopsin showing the pocket where retinal resides. Hydrophobic residues are colored yellow. Red marks the C-terminal end of the protein. All other residues are colored blue.
Proton transfer is described in Luecke, et al. [5] Refer to Figure 3.4. The retinal is linked to bacteriorhodopsin through residue Lys216 (green). The steps of proton transfer are:
Figure 3.4. Side view of bacteriorhodopsin showing residues involved with proton transfer: Asp85 (magenta) and Asp96 (orange). Lys216 is marked with green and is the residue to which the retinal is attached. Hydrophobic residues are colored yellow. Red marks the C-terminal end of the protein. All other residues are colored blue.
[1] R. Sayle and E.J. Milner-White. RasMol: Biomolecular graphics for all. Trends in Biochemical Sciences (TIBS), September 1995, Vol. 20, No. 9, p. 374.
[2] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242 (2000).
[3] R. MacKinnon. Potassium channels. FEBS Letters. 2003 Nov 27;555(1):62-5.
[4] R. Henderson, J.M. Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann, K.H. Downing. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899-929.
[5] H. Luecke, B. Schobert, H-T. Richter, J-P. Cartailler, J.K. Lanyi. Structural Changes in Bacteriorhodopsin During Ion Transport at 2 Angstrom Resolution. Science, Vol 286, Issue 5438, 255-260 , 8 October 1999.