The story of a Foldit design
As discussed in previous blog posts, some Foldit player solutions from design puzzles are chosen for synthesis in the Baker Lab. One design in particular, from Puzzle 854, has recently yielded some promising results in the wet lab. Below, we follow this design's journey from video game to test tube:
1. Once we select a design to be synthesized in the lab, we extract the amino acid sequence from the design and reverse transcribe this into a sequence of DNA bases (i.e. a gene), adding a special tag that will come in handy later.
2. We order this gene as a DNA molecule from a gene synthesis company, and splice the gene into a larger, circular piece of DNA called a plasmid. The plasmid now containing our gene is inserted into E. coli bacteria, which we allow to grow and reproduce in an incubator. These bacteria will transcribe and translate our gene as if it were one of their own, producing our design as a polypeptide chain; if the protein design is good, the polypeptide chain will naturally fold up into the design structure.
3. Once the bacteria have grown to saturation and produced a large amount of our protein, we break open the bacteria cells and separate our protein from the other bits of E. coli using the special tag we added in step 1. At this stage, we can see whether the bacteria were able to produce our protein and whether the protein is soluble. Unstructured proteins will usually be degraded by the E. coli or otherwise form insoluble aggregates.
SDS-PAGE. A mixed protein sample (stained blue) is passed through a polyacrylamide gel from top to bottom, such that smaller proteins travel faster through the gel. Here, three samples are shown: all soluble proteins from a bacterial cell (left), proteins lacking the special tag we added in step 1 (middle), and proteins with the special tag (right). Although the first two samples have many bands (different proteins) spread across the length of the gel, the sample on the right is dominated by a single large blot (our protein) near the bottom of the gel.
4. We use size-exclusion chromatography (SEC), which separates proteins based on their size, to get rid of other protein impurities. This step also gives us information about the oligomeric state of our protein (unstable proteins with exposed hydrophobic residues tend to self-associate into oligomers). Structured monomers will behave differently on the column than oligomers or unstructured aggregates.
SEC Trace. Proteins are passed through a matrix such that larger proteins travel faster through the matrix and are collected sooner (at the left end of the x-axis); absorbance of UV light is used to measure the protein concentration (y-axis) of samples as they are collected. You can see that in the case of our protein, this step was hardly necessary because the protein is unusually pure, evidenced by a single dominant peak at 14 mL. Furthermore, the placement of the peak at 14 mL corresponds precisely to the expected size of the design, indicating the protein is monomeric.
5. After we have purified our protein, we can use circular dichroism (CD) to measure its secondary structure content. This technique measures a protein's absorption of circularly-polarized light and can tell us about the amount of α-helix or β-sheet in the protein. This measure also allows us to monitor how the protein unfolds when we raise the temperature.
Circular Dichroism. Different elements of protein structure interact differently with circularly-polarized light. At 25°C (blue trace, top) our protein shows a CD profile characteristic of a protein with a large α-helical content. At 95°C (red trace, top), the shape of the profile is less pronounced, indicating a loss of secondary structure; the secondary structure is recovered upon cooling back to 25°C (green trace, top). The bottom trace shows how the CD signal at 220 nm changes as we raise the temperature of the protein sample. The gradual, broad slope of this trace indicates noncooperative, multi-state unfolding.
The protein described above is being prepared for crystallization. If successful, we may be able to obtain a high-resolution crystal structure of the protein and make a comparison with the designed structure. Please note that we are continuing to work with other proteins that may be less well-behaved, and hope to order new designs soon!
Check out the latest design puzzle here!( Posted by bkoep 80 1005 | Wed, 06/18/2014 - 00:25 | 3 comments )