DIY Molecular Biology
Did you know that you can be a molecular biologist at home? There are public databases loaded with information on analysis for humans and a wide variety of animals, plants, and microbes that have been sequenced for decades and in laboratories around the world.
You can access them! You can visualize them! You can even see how segments of a gene are transcribed and translated and folded into a protein structure! All the isolation and sequencing work has been done for you (thank goodness), and you are free to read and visualize the code that makes an organism what it is.
All life on earth relies on this 4 letter code, and we share a huge portion of our genomes with every other lifeform on the planet. Often you can look at an animal and assign its species by the recognizable characteristics. But what if they look exactly alike? How do scientists discover "cryptic species" that look identical but are not actually the same? It all comes down to genomic analysis. Even tiny differences can make a huge difference when it comes to DNA.
Let's play around with the publicly available resources.
Here's an example using a venom
protein from the Australian king brown snake (Pseudechis australis) also known as the mulga snake.
This snake is found all over Australia, and while its venom is not the most potent of Australia's snakes, they can be very dangerous. A high dose of venom can have neurotoxic effects, cause paralysis from muscle damage, and affect blood clotting.
Basic Overview of How to Visualize and Investigate Protein Molecules
1) Pick your protein of interest.
Cysteine-rich secretory proteins (CRISPs) are hypothesized to play important roles in reptile venom, where they act by blocking cyclic nucleotide-gated and voltage-gated ion channels and inhibiting smooth muscle contraction (i.e. neurotoxins as in many Elapid snakes)
Example = Australian king brown snake (Pseudechis australis) cyclic nucleotide-gated (CNG) ion channel blockers called pseudechetoxins (PsTx)
2) Find the nucleotide sequence in GenBank (www.ncbi.nlm.nih.gov/genbank)
3) Select “Nucleotide” in search bar and search “pseudechetoxin”
a) Note that there are many species who have this gene
4) To find our specific species’ venom protein, search for “pseudechetoxin Pseudechis australis” to get the sequence for the species we have chosen
5) The top result is the mRNA sequence for the Pseudechis australis pseudechetoxin and it is 1319 base pairs long
6) Open the mRNA sequence to see the studies that determined it and submitted it, the full nucleotide sequence, and the amino acid translation.
Note that the CDS (coding sequence) is only from base pairs 67 – 783
This means that the amino acid sequence only reflects this portion of the sequence given and that the protein will be translated from these
Because proteins use a triplet codon for translation, there are 238 amino acids from the 716 bases in the CDS
7) Through experimentation in determined that that three specific amino acids at particular sites (Lysine at site 167, Lysine at 174 and Arginine at 175) increase binding to the olfactory CNG (CNGA2) ion channel.
At the right side of the GenBank page, click “Protein”
Note that the PDB ID is 2DDA
8) Go to PDB (protein databank) at http://www.rcsb.org and search “2DDA”
You can see the molecular 3D model of the protein with the secondary structures (alpha helices and beta sheets) folded into the tertiary structure with loops and folds:
The page also includes more in-depth information about the protein, including the ligands that can bind to it, and how tightly they will bind
If you select “3D view” at the top, you can spin and examine the molecule more closely
Select “ligand view” to select a particular ligand from the drop down and see the molecular interactions (steric interactions, hydrogen bonds, and Van der Waals forces)
There are 4 chains to this protein, but the one that works as a neurotoxin by binding ion channels is the 211 amino acid long A chain.
Is that amazing? You can pick any gene you want, play around with the database and see what is available to you.