Tutorial developed by Ross Feldberg, Dept. of Biology, Tufts University
The Ras protein is a monomeric globular protein of 189 amino acids (21 kDa molecular mass) which is associated with the plasma membrane and which binds either GDP or GTP. Ras acts as a molecular switch. When Ras contains bound GDP it is in the resting or off position and is "inactive". In response to exposure of the cell to certain growth promoting stimuli, Ras is induced to exchange its bound GDP for a GTP. With GTP bound, Ras is "switched on" and is able to interact with and activate other proteins (its "downstream targets"). In biological process, any on switch must be balanced by an off switch. In the case of Ras, the protein itself has a very low intrinsic ability to hydrolyze GTP back to GDP, thus turning itself into the off state (like a light switch on a timer). However, in the case of Ras this intrinsic activity is quite low and switching Ras off requires extrinsic proteins termed GTPase-activating proteins (GAPs) that interact with Ras and greatly accelerate the conversion of GTP to GDP. Any mutation in Ras which affects its ability to interact with GAP or to convert GTP back to GDP will result in a prolonged activation of the protein and consequently a prolonged signal to the cell telling it to continue to grow and divide.
This is a relatively small protein (177 amino acids) and the guanine nucleotide bound to the protein is shown as spacefilling to give you a sense of the relative size of a globular protein and a cofactor or substrate. Wireframe representation allows us to pick out every amino acid, but it is inaccurate. Here we switch from wireframe to spacefilling representation showing the van der Waals radii of the individual atoms (minus hydrogens). This gives a more accurate protrayal of how the protein would be "seen" by other molecules (i.e. if they approach closer than the van der Waals radii, strong repulsive forces are exerted). This is a globular protein - the polypeptide chain folds back on itself to form a relatively compact structure. The guanine nucleotide is displayed in magenta to make it stand out. For a molecule this complicated, it is difficult to extract useful information from either the wireframe or spacefilling representation.
Globular proteins are composed of distinct secondary structural
elements folded back on one another to achieve an overall compact shape. To better visualize
this we can eliminate all amino acid side chains from our representation and focus on the
polypeptide backbone (-NH-Calpha-CO-). To emphasize secondary structure we use the
cartoon representation in which beta strands are yellow arrows (arrowhead is at the C
terminal end of each strand), alpha helices are red coils. Also, polypepide regions with no
regular secondary structure are white ropes and beta turns are shown in blue. Take a minute
to rotate this structure.
What is
the relationship of the beta strands to one another? What is the relationship of the helical
regions to the beta strands? Rotate this molecule and note that only a relatively small
portion of the protein is actually involved in binding the nucleotide. However, those
regions will define the specificity of the protein. Finally, use the Chime menu to determine
where the cysteine residues are located and if they are close enough together to form
disulfide bonds.
The specific regions of the ras protein found to undergo a conformational change upon hydrolysis of GTP to GDP are shown here in purple. Note that much of the switch region is not in close contact with the nucleotide. Which amino acids are involved in the switch regions? What might you propose is the function of the conformational change in this region?
Proteins fold to reach a minimum energy level. In general, this is accomplished when amino acids that are hydrophobic are brought together to face one another in the interior of a protein and polar or charged hydrophilic amino acids face the surrounding solvent. However, this rule is not absolute. The animation shows the hydrophobic residues in yellow and hydrophilic amino acids as blue. If you rotate the molecule, you will note that for much of the protein, blue covers yellow and yellow is only visible at the bottom of specific channels in the protein. This is not always the case since regions of a protein involved in interactions with other proteins may be hydrophobic and lie at the surface of the protein.
When a stretch of alpha helix runs along the surface of a protein, the side facing the protein is often made up of hydrophobic side chains, while the side facing the solvent is made of hydrophilic residues. A helix in which one side is mainly hydrophobic and the other mainly hydrophilic is termed an " Amphipathic Helix". The helix that extends from residue 155-164 (AFVLVIEYTRER) is an amphipathic helix and here the hydrophobic residues are colored yellow while the polar residues are blue. How many amino acids separate residues that face along one surface of the helix? Can you imagine a situation where the amino acids that project away from the protein would be hydrophobic?
Naturally occuring mutations at ras residues 12, 13, 59 and 61 result in uncontrolled cell growth with gly12 or gln61 being responsible in the majority of the cases. However, close inspection of these residues raised interesting questions. Gln61 is too far away from the GTP to directly act as the attacking nucleophile and gly12 with its R group of H would not be expected to play any direct role in catalysis. The answer to this puzzle was only found when it was realized that an accessory protein, GAP (GTPase Activating Protein) was crucial for hydrolyis of the GTP to GDP. (see next animation)
Ras protein has a very inefficient GTPase activity by itself, but it interacts with another protein, GAP, which enhances GTP hydrolysis many orders of magnitude. The full GAP protein is 1047 amino acids, but the portion shown associated with ras only covers residues 714-1047 (334 amino acids). This is the catalytically active portion of the protein. The action of GAP protein is dependent on a loop that includes GAP-arg789 and which protrudes into the Ras protein and interacts with the beta phosphate of GTP. Ras-gln61 appears to also play a direct role in the transition state. Although it is too far away from the GTP to be the nucleophile, gln61 acts to activate a water molecule which is the initial nucleophile. Finally, Gly12 with its hydrogen atom R group plays no direct role in the catalysis, but occupies a position close the GAP arg loop. Substitution of any other amino acid at this location results in steric interactions which prevent the GAP protein from assuming the correct structure for catalysis.
This tutorial was developed by Ross S. Feldberg (Dept. of Biology,
Tufts University) with help from a Teaching with Technology grant from the Center for
Teaching Excellence at Tufts.
Any corrections or comments should be sent to rfeldber@tufts.edu