New anti-Ebola puzzle posted!
The results from the last two anti-Ebola peptide design puzzles have been very good. In particular, some of the new topologies and disulfide connectivity patterns that players have found have been quite interesting. I have, however, noticed that a lot of designs had some large voids, particularly between the peptide and the glycoprotein. This might be in part because the starting configuration of the peptide (which we provided) wasn't great -- but that just means we need to try again! We're posting a new anti-Ebola peptide design puzzle for you to try, this time with two starting configurations that might lend themselves a bit better to the creation of a void-free peptide-protein interface. Good luck!( Posted by v_mulligan 126 1119 | Thu, 09/04/2014 - 22:37 | 0 comments )
New anti-Ebola puzzle posted
Based on player feedback, we've posted another design puzzle to create a peptide inhibitor of the Ebola glycoprotein, this one with 30 amino acid residues. The puzzle is live now, and expires just before the scheduled server downtime on Sept. 3rd. Bonne chance!( Posted by v_mulligan 126 1119 | Wed, 08/27/2014 - 21:26 | 0 comments )
Continuing the battle against Ebola
As you know, the current Ebola outbreak in Western Africa has now claimed over 1,000 lives, making it the worst Ebola outbreak to date. Currently, no proven treatment or vaccine exists to combat Ebola. It has been some time since our last Ebola design puzzles (http://fold.it/portal/node/997612 and https://fold.it/portal/node/997525) were posted. These, in combination with the initial hotspot-finding puzzle (https://fold.it/portal/node/996919), have both yielded some great leads that we are currently working to test in the wet lab. In particular, some of the best player hotspots yielded excellent starting points for the design of small, cyclic peptides that we're quite excited about.
The process of going from a candidate design to a drug ready for deployment in the field is a long one. We start by screening a large number of candidates for binding to the Ebola surface glycoprotein, using high-throughput methods. Because the Ebola virus is obviously quite dangerous, we can't work with it ourselves in our wet lab. We therefore use Ebola proteins that have been made in harmless strains of bacteria, using recombinant DNA technology. As for the candidate designs, we express these in baker's yeast, also using recombinant techniques. Because we are using high-throughput methods at this stage, we expect some false positives from our screen. Some candidates, for example, are just generally "sticky", and bind to pretty much anything. These would not be useful as drugs, since they'd stick to all sorts of other things in the body, but they show up in the initial screen as a hit.
Once we have some possible hits from our initial screen, we express and purify larger amounts of these second-round candidates for more careful experimental validation using lower-throughput techniques. At this stage, we need to do careful controls to confirm that the second-round candidates aren't just generally sticky. We also start to get quantitative at this point, to see whether a design binds tightly enough to be a useful drug, or whether it needs further redesign to improve its binding affinity.
Through collaborators, we will ultimately test candidates that pass all of our tests in cell culture, then in animals infected with the virus. The road to human trials is longer still, since it's necessary to show that a drug is safe before it can be given to sick people. Even with a disease as horrible as Ebola, one has to be sure that the treatment isn't worse than the disease. We're not yet anywhere near the stage at which candidates could be administered to people suffering from the Ebola virus -- but with time, effort, and patience, we hope to get there.
In the mean time, now that CASP is winding down, we will be posting a few more Ebola puzzles in the next few days. In particular, I'm curious about whether players could do better than the automated algorithms at designing a heavily disulfide cross-linked peptide to bind to the Ebola glycoprotein... We look forward to finding out!( Posted by v_mulligan 126 1119 | Thu, 08/14/2014 - 04:32 | 0 comments )
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 126 1237 | Wed, 06/18/2014 - 00:25 | 3 comments )