Microbiology Stuff

Tomorrow is my microbiology lab final. For the past couple weeks I've been memorizing 12 microorganisms and how they test in 12 different tests. Plus a few others. Here's some fun devices I've come up with for memorizing the activity of these particular organisms in these particular tests.

These are the culprits.

The first four tests we're going to talk about is the IMVC. The indole test tests for the ability of organisms to convert W into indole via reductive deamination. The final products you get are indole, pyruvic acid and ammonia. A positive indole gives you a red/violet color and a negative gives you yellow. 

The methyl red test is used to identify bacteria that produce stable acid end products by mixed acid fermentation of glucose. This includes enterics that metabolize pyruvic acid to other acids. When the pH of the medium is lowered, it turns the medium red. Neutral end products would give you a pH of about six with a yellow color.

The voges proskauer (sp?) is used to detect organisms that produce acetoin using the butanediol pathway. The color changes to red in a positive test and is yellow/brown in negative.

The simmons citrate agar has sodium nitrate as the only carbon source and ammonium phosphate as a nitrogen source. There's blue dye (that's green at pH 6.9, blue at 7.6) involved. The bacteria that survive in the medium and utilize citrate also convert ammonium phosphate to ammonia, making it alkaline. As the pH goes up the color changes from green to blue. A positive citrate is indicated by a blue color.

I memorized these twelves organisms' activity in IMVC by treating it as the group of testing that it is. You can first remember that in IMVC, E. coli and Shigella spp. both test as ++--

You then need to remember that Enterobacter cloacae tests the opposite of E. coli, along with two others. The way you remember the three that test as --++ is by calling them "mar-cl-polo" as in the photo above.

P. aeruginosa is the only one for IMVC that tests as ---+

The two proteus' test the same for IMVC with the exception of the citrate test.

And then I realized that P. mirabilis along with two others has the same pattern. The way that I memorized these three together is kind of silly, so I won't say it.

Then these two ^^ are the same for indole and citrate but switched for methyl red and VP.

Now, I also wanted to individually memorize all the tests individually. So I chose to memorize the +'s for some and -'s for others...when you're doing this you better make damn well sure that you know which ones you memorized the +'s for and which ones you memorized the -'s for. 

I'm particularly proud of this one. VP+ is almost the same thing as MeRed -. 

For VP, I came up with "Very pleased 2 know Ser. Marc E. Clar" as a device, and for the MeRed - I just remember to add aeruginosa to the list.

Couldn't really think of a good way to memorize simmons citrate...

The two decarboxylate tests were also kinda tricky. So for K-Decarboxylase (lysine...man I'm so glad I am taking biochem at the same time, it makes everything make more sense) I remember that since the letter for Lys is K, it has the 2 Klebsiella's and then their friends marc and sal. This test determines if a bacterium can use lysine as a carbon source. When it turns from yellow to purple, it's positive.

Cl a mm just made sense for this one to me. I don't know why I memorized it that easily. This determines if a bacterium can use ornithine as a carbon source. If so, there'll be an increase in pH and a change in color. At a low pH, it'll be yellow and it'll be purple in basic conditions. Positive is purple.

This one I only came up with tonight. It's a shitty one but it's all I got. For F (phenylalanine)-Deaminase...

"For dad: 2 Props: Salmon and Steak"

F-Deaminase: 2 Proteus', Salmonella, Stuartii

This determines if an organism can make F-Deaminase, needed to use phenylalanine as a carbon source. If it's positive, a product combines with iron to produce a green color.

The Urease I had to just kind of memorize, no fun here. The urease test determines if a bacteria can make the exoenzyme urease to hyrdolyze urea to ammonia and carbon dioxide. It'll come up as red in an acidic environment.

H2S tests for the reduction to gas. Either cysteine desulfurase will reduce cysteine to pyruvate or thiosulfate reductase catalyzes the reduction of sulfur in the electron transfer chain. In SIM medium, H2S gas combines with iron to form a black precipitate.

Since almost all the organisms are motile, I decided to memorize the three that aren't.

And I chose to memorize lac+ and suc- fermentors 

There will be more later on activity in agars (HE, EMB, MacConkey, etc) and some other stuff, but for now, the doge needs walking.

The snowy doge.

Protein folding and other things that are nice.

There's reasons for why proteins go from nice long strands of stuff to more compact folded things. The first part of it is conformational entropy... The -deltaS in this case makes a positive contribution because you're going from a very random thing to a much less random thing, which means entropy is technically working against folding.

But despite that, the thing still folds. That means deltaG must still be negative. So that means this happens because deltaH must be very negative, which comes from the nice interactions that happen between the amino acid side chains in the folded thing. 

Another thing going on in protein folding is charge-charge interactions. This comes from charged side chains that might form bridges. However, if pH changes, the side chains might lose their charge, breaking the ionic bonding between them, which would then contribute to denaturing the folded thing.

The next factor to look at is internal hydrogen bonding between side chain. Side chains with OH, NH2, etc are the ones that can participate in non-covalent *technically* bonding interactions.

If we go back far enough in our biochemistry memory map, we can maybe remember that neutral-neutral interactions (induced dipoles) are the strongest if the interaction is physically close enough, which is what I guess happens here. The non polar groups in the protein, when they pack in densely enough, will contribute to a more stable protein. The folding up of the protein also reduces any favorable interactions that the chains may have with solvent...the unfolded chain may have residues that CAN interact nicely with water.

So, looking at the enthalpy changes going from unfolded to folded, we see that the type of non covalent bonding changes..you go from interactions between the open chain and the solvent to interactions within the chain.

*Sort of * along those lines, there's disulfide bonds. They're usually the only covalent bonds that are going to form when a protein folds up, and the sentence is BLUE is very important because although disulfide bonds contribute to enthalpy, they have more entropic effects.

Final consideration for us is the hydrophobic effect. Myoglobin is actually stabilized by it.

When you have hydrophobic residues in contact with the solvent in open chain form, you get those CLATHRATES that we talked about in chapter...3? 2? oh, who remembers?

Anyway, when you form clathrates, water gets ordered around stuff that it doesn't really want to be around, which is bad for entropy. 

So when you have an unfolded protein with lots of crappy clathrates going to a folded thing where all the hydrophobic shit is inside...I'd say that frees up some of those unhappy water molecules. That's pretty good times.

Disulfide bonds are like the one girlfriend you had in high school..you're going to keep talking about her. In Bovine pancreatic trypsin inhibitor, we have three of them. BPTI is pretty hard to denature but putting it in really acidic stuff at temperatures above 100degreesC is going to get the job done. Eventually.

HOWEVER. If you cleave just ONE of those little suckers, you can start denaturing that shit at like...

...a much lower temperature !

And if you reduce all THREE of the disulfide bond, that shit is going to fall apart at room temperature.

In short, if you have disulfide bonds, you've got less conformational entropy in your unfolded state than if you don't have disulfide bonds....there's just less shapes for you to be in when you're all connected like that. Disulfide bonds are typically found in proteins that get taken out of the cell (RNase, BPTI, Insulin...)... In the cell you've got a reducing environment ... which...HELLO? Would reduce that shit. But outside the cell, you've got an oxidizing environment which is actually going to help stabilize those -S-S-'s

Chaperones keep proteins from getting into trouble..

Like this mess of an example..

A summary of what happens.

A short summary slide to help you remember which amino acids are going to be predominantly present in which things. I'm especially proud of the vampires.

Some thoughts and then some better formed thoughts.

Before I start talking about further details of protein folding and protein structure, I want to go over a few things (with myself since no one is listening).

Figuring out the pattern of this was kind of annoying, but I had Lenny look at this picture before I decided to post it to ensure 100% that this was accurate. This is a recipe for charging and translating a 5-peptide long thing. You charge the suckers, you put the first charged fmet into the psite, then 8 more for the next four and the four times that the ribosome moves over, and then the last one for releasing the chain.

In the spirit of charging tRNA, let's discuss this one more time, with gusto. Your Amino acid is going to attack an ATP to form an aminacyl adenylate. This is the first important step to make the amino acid more reactive.

Your tRNA's 3' end has a ribose with an adenosine and 2 OH's. One of them will attack the aminoacyl adenylate at the amino acid's carboxy carbon. That'll give you two classes of products that we talked about already.

That's just a slightly better look at what is doing the attacking here.

A bit about the ribsome. It's made out of RNA and proteins. The rRNA has self-complementary sequences and an active site. The active site is not in a protein. This is what gives the rRNA special "ribozyme" function.

This is the initiation complex. The IF2 with it's GTP friend is at the shine delgarno sequence, the first amino acid is at the AUG and IF1,3 are bound to the 30s subunit of the ribosome.

When the initiation complex attaches to the 50s ribosome subunit, you get the 70s complex with the mRNA chilling and waiting for translation to start. At the beginning, you've got your first tRNA with FMet in the P site, and the E and A sites are empty.

Peptidyl transferase is where the magic happens to form peptide bonds. Those are the things that form between the nucleophilic deprotonated amino group of the amino acid at the A site that just came in and the carboxy carbon of the tRNA of the P site.

When there's a stop codon, there's no tRNA to recognize it, so releasing factors bind at or near the A site.

Termination ends with peptidyl transferase using water as a nucleophile for polypeptide hydrolysis.

This is in the spirit of all those lovely drugs that interfere in one way or another with translation. Aspirin is different. this one has covalent modification.

Now that those words are over with, I want to start finally talking about some shit dealing with structure for realsies.

While the primary structure of protein is the amino acids, the secondary is the stuffy shapes that come out...usually beta helices or alpha sheets. Alpha helices are right handed, with stabilization from hydrogen bonding between N-H---C=O and the hydrogen bonding is nearly parallel to the helix. R groups of the amino acids point out of the helix. The two termini (N and C) give the alpha helix dipolar character.

That there is a horrible rendering of beta sheets. Beta sheets have hydrogen bonding between adjacent chains...sort of like what I drew but better. The hydrogen bonds are perpendicular to the chains...which I sort of depicted. 

The types of beta sheets. I'd say we're more friendly/happy about antiparallel beta sheets. 

I'm going to entirely butcher the description of this, but I'll give it a shot. So..this depicts a peptide chain of a bunch of amino acids. That's the R groups there. Different structures of proteins have different hydrogen bonding between the C=O and the H-N. The number of atoms away the two that are hydrogen bonding describes the type of helix you get. So..the alpha helix has 13 atoms in the "loop" of the hydrogen bond, while the 3_10 helix is bound 10 atoms away. So on.

Another type of helix. This one is funky. It's left handed, and a third of the stuff in it is proline. 

I spelled aneurysm wrong. Yep.

So these angles...from the alpha carbon toward the end of the c and n termini....are important.

Beta strands have nice large angles. In the three digits. Because beta strands are spread out and wide. Alpha helices have smaller angles. Because they're tighter, like those things that bounce up and down stairs that I can't think of the word for right now. Jolly somethings?

This is a mess. I'm sorry. If you don't take biochem, I am too braintired to explain this for real. Basically it's a plot of what happens to be the allowed angles for the phi and psi angles. Things fall into certain places.

Now that that mess is over with, we can talk more about something I actually really like.

Those are the amino acids that are most prevalent in those types of fibrous proteins. I sort of have pneumonic things for memorizing them but they mostly deal with peoples names who I know ( like the doctors I work with), so that wouldn't be helpful to mention to anyone but me.

Fibroin's most prevalent amino acids. 

Okay, there's sort of a reliable way to memorize the three most prevalent ones in collagen. Collagen sounds like College and your GPA in college is important. Glycine, Proline, Alanine are important in collagen. Hah. 

Elastin has the three most ordinary ones that are most prevalent in the structure.

Alpha keratins have right handed helices that twist around each other, with every third/fourth amino acid having a non-polar/hydrophobic side chain. There's non-covalent bonding to stabilize the helices by hydrophobic interactions.

The disulfide bonds are pretty important, too.

The second "type" of keratins.

Fibroins have lots of b-sheet structure, with long regions of anti-parallel sheets with mostly glycine and alanine. Glycine is every other residue followed by A or S...so it looks like ... GAGAGSGAGSGA ish. Something like that.

In fibroin, bonding between sheets occurs via weak van der waals interactions between side chains. But not all of fibroin is beta sheet, as it has some bulky stuff up in there inside folded regions that let the stuff be stretchyish.

Collagen is my favorite. It is a triple helix of three peptide chains which are all individually left-handed but warp around each other in a right-handed fashion. There's hydrogen bonding between the chains and the pattern here is G-X-Y with every third residue near the center being glycine.

A little while ago, couple posts maybe, I promised to talk about when amino acids get hydroxylated in post-translational modification or some shit like that. This is that time that I'm going to talk about this. Collagen has a lot of this. Collagen has a lot of Glycine, Proline, Alanine, and a lot of the prolines get hydroxylated when collagen is being made.

This also happens to the lysines in collagen.

For the hydroxylation, you need vitamin C (L-ascorbic acid). When you don't have this, you get scurvy. Apparently someone I know through someone else got scurvy, which is kind of crazy. Scurvy is basically weakening of these awesome collagen fibers because there's no hydroxylation occurring in the P's and K's. More more more:

I get so excited when organic chemistry is mentioned. Lysine side chains get oxidized to aldehyde derivatives. Those derivatives can then interact with other either derivatives or with actual Lysine residues via aldol condensation and dehydration to form these crosslinks. As you get old and stupid (or just older), this shit builds up over time which is no good.

The way that collagen is made is pretty fucking sweet. You've got those ribosomes spitting out the peptide chain. Then you've got hydroxylation of P's and K's, giving you Preprocollagen that leaves the endoplasmic reticulum, where it's being made.

Out in the cytoplasm, you've got these sugars added to give you procollagen. There's also some weird stuff going on at the N and C terminals, they don't have the normal structures of collagen fiber.

This is kind of what starts happening before it leaves the cell membrane.

Outside the cell, the terminal regions get chopped off by proteases and then the lysine side chains get deaminated to form those crosslinks we had up there.

Elastin is also pretty beautiful. There's also lysine side chains involved in crosslinking, but much differently from collagen and a lot fewer of them.

That's a nice (if I may say so myself...) summary of the four fibrous protein structure things.

I don't want to get too messy with these posts, I feel like they're messy. So I'm going to stop for now and talk about globular proteins and more about folding patterns in the next post.