Tryptophan

               I love turkey.  I love cranberry sauce that has the ridges from its can.  Most of all, I love stuffing.  Thanksgiving is a great food holiday!

                I bet we’ve all seen family members taking a Thanksgiving snooze after eating too much turkey.  (I’m sure Uncle Dave’s impromptu nap has nothing to do with the number of beers drunk while lying on the couch watching football…)  Many people feel that sleepiness following large Thanksgiving meals is due to the tryptophan found in the turkey.  A quick PubMed search of “tryptophan and sleep” will turn up 648 results.  Tryptophan does play a role in sleep.  However, tryptophan isn’t only found in turkey – it’s in egg, soybeans, cheddar cheese, chicken, beef, salmon and even bananas!  According to Snopes.com, you will not eat enough tryptophan on Thanksgiving to feel any appreciable effects, much like every other day of your life when you are ingesting many other foods that contain the molecule.  Also, most experts agree that the post-dinner fatigue is probably due to both an increase in metabolism and blood flow to your gastrointestinal tract.

                So… what is this little molecule?  What does it look like?  What else do we know about it?

                Figure 45.1 shows you what tryptophan looks like.  It’s a small molecule of carbon, nitrogen, hydrogen, and oxygen.  Most things in your body are made of the same four elements (although sometimes we throw in sulfur, phosphorous or a metal for fun).  You’ll notice that the left end of the molecule has two rings of atoms.  These rings are cool because it allows tryptophan to be fluorescent!  If you shine light of 280 nm on tryptophan, it will spit light back at you ranging from 300 – 350 nm.  

                Tryptophan is also something that you’ve heard me talk about in these posts before: it is an amino acid.  In fact, tryptophan is an essential amino acid.  What does that mean?  It means our body has no way of making it, so we must bring it into our bodies via our diets.  Our body needs amino acids to create proteins (oh, that pesky Central Dogma post just never goes away, does it?).  Of the 20 amino acid used to build proteins, 9 are essential.  We have the tools (enzymes, precursors) present in our cells to make all the non-essential amino acids, but essential ones must be ingested.  They are “essential” to our diet, if you will.  Without these amino acids, our bodies won’t make proteins properly and, subsequently, won’t run properly.  Do you remember Jurassic Park?  The dinosaurs were all unable to synthesize lysine (another amino acid).  Unless the animals’ diets were supplemented with lysine, they would die.  

                Interestingly, our bodies do have the tools to take tryptophan and turn it into other useful things.  For example, tryptophan will bind to the enzyme tryptophan hydroxylase and out will pop serotonin.  Serotonin is a neurotransmitter that is commonly thought to “make people feel happy.” 

                Serotonin can then, in turn, bind to N-acetyltransferase and be converted into another molecule that will bind to 5-hydroxyindole-o-methyltransferase and become melatonin.  This molecule is involved in our circadian rhythms.

                I will come back to tryptophan in the last post of the “From DNA to Protein” series, which will be posted soon.

                Until then...

Essential amino acid: Those amino acids that must be provided by our diets

Non-essential amino acid: those amino acids that our body can make

REFERENCES

http://www.snopes.com/food/ingredient/turkey.asp

Schaechter, JD and Wurtmann, RJ.  “Serotonin release varies with brain tryptophan levels.” (1990) Brain Research 532 (1-2) pgs 203 – 210

Wurtman, RJ and Anton-Tay, F. “The mammalian pineal as a neuroendocrine transducer.” Recent Prog. Horm. Res. 25, pgs 493 – 522.

From DNA to Protein, Step 2

It’s time to move on to Step Two!  (Again, if you want more background on this post, see the earlier posts in this series and/or The Central Dogma!)

Let’s remember what we did in Step One:  Proteins are strings of amino acids.  Cells are much better at making proteins than any kind of human techniques available.  Scientists use bacteria to make the protein they want.  Bacteria need the instructions to make protein.  Instructions come in the form of DNA.  The end of Step One was a circular piece of DNA, called a plasmid, which contained the DNA that encoded the protein we wanted.  We put that little piece of DNA into a couple of bacteria.

Now what?

BACKGROUND/EASY EXPLANATION

                One small bacterium is not enough to make all the protein we need. 

                To offer some perspective:

                Let’s say you need 50 ug (Microscopes and Photography post) of the protein GST-RED to perform one experiment.

                One protein molecule of GST-RED weighs ~ 0.00000000000000498 ug.  For real.  See how small that is!?  Even if that one little bacterium pumped out 1000 protein molecules, you’d only get ~ 0.00000000000498 ug.  (You are free to count my zeros.  All I really want you to take away from this is how little a protein weighs versus how much is needed to do experiments.)

                How do you make a lot of protein then?  Well, you make a lot of bacteria!

                E. coli bacteria are quite happy to grow in a liquid called lysogeny broth (also known as Luria Broth or simply “LB,” among other things).  It’s a pale yellow liquid chock full of food that bacteria love.  It comes in a powdered form (or you can make it from scratch with laboratory supplies).  You weigh out the proper amount, add water and sterilize it.  The sterilization kills any bacteria that may be living in the liquid or happen upon the liquid before you’re ready for it.  This ensures that once you add your E. coli bacteria that are bearing your plasmid DNA, all the bacteria that will subsequently grow in the LB will also be E. coli bearing your plasmid DNA.

                How much LB do we usually make?  It depends.  It can be as small 1 liter or as large as 24 liters.  Once, I made 85 liters.

               Again, perspective:  For some proteins, 1 liter of bacteria will give you 10000 ug of protein.  See why growing more bacteria is so useful?

                Let’s be nice and say you only need 1 liter.  You have your 1 liter flask of LB.  To it, you add E. coli that has your plasmid DNA.  Then, you place the flask in an incubator at 37°C (98°F) and gently shake it.  The shaking helps the bacteria grow.  

                And you wait. 

The bacteria are so happy with all the food around that they start to multiply.  Usually, they can double their numbers in about twenty minutes.  

As more and more bacteria start to occupy the flask, the once clear yellow liquid starts to become very cloudy.  Very quickly, we have millions of bacteria in a flask that are churning out our protein.  All those cells are jam-packed with GST-RED.  

Like our refrigerators, only a finite amount of food can exist in a particular area.  We don’t want to overgrow the bacteria to the point where they run out of food and start to die.  For this reason, we stop growing the bacteria at a certain point by removing them from the warm temperature and centrifuging (Tales From the Bench post) the liquid + bacteria mixture.  Centrifuging serves to separate the bacteria from the LB.  We are left with a pellet of bacteria that goes in the freezer until Step 3!

If you now refer back to Figure 42.1, you’ll see that I depicted the end of Step 2 as individual bacteria molecules (rounded rectangles) that are full of our protein (red triangle!)

Step Three, the last step!, is how we get it out of the bacteria!

MORE INFORMATION / INTERMEDIATE AND DIFFICULT EXPLANATIONS

Controlling protein expression.  The plasmid DNA scientists use is actually quite sophisticated.  For DNA to make protein, a molecule called RNA polymerase must bind to the DNA ahead of the gene.  When you only have a few bacteria, it’s not necessary to start making protein.  Sometimes the protein you want to make is toxic to the cells or adversely affects their growth.  It is to the scientists benefit to first grow all the bacteria they need and then have them make protein for a short time.  The plasmids allow us to do this because a protein actually binds to the DNA where the RNA polymerase needs to bind.  It blocks the gene from being read.  Once we have enough bacteria, we add a chemical to the bacteria that removes the blocking protein and allows RNA polymerase to bind and protein to be made.

Ensuring plasmid DNA is in all the bacteria.  In the last post, I alluded to the fact that bacteria can spit out plasmids if they have no use for them.  This isn’t good for scientists.  We need the plasmid DNA to stay in there so we get protein.  Again, the plasmids are sophisticated.  Another gene exists in the plasmid that encodes a protein involved in antibiotic resistance.  This gene is never blocked like our protein gene is.  If the plasmid is in there, the bacteria are making this protein which renders the bacteria immune to a particular antibiotic.  In our LB, we put a small amount of antibiotic.  This serves to kills all bacteria except those bearing the plasmid.

                Some finer points exist in this step, but they are minor.  If you’d like to know how scientists judge how cloudy their LB is (although we can, many of us don’t simply use our eyes) or what exactly the chemicals/antibiotics we use are, just ask!

REFERENCES

Me, myself, and I.

From DNA to Protein, Step 1

                 Okay!  We’re going to do it!  We’re going to make a lot of protein and then purify it.  Are you ready?

                Let’s start with a few clarifications first.

One.  I highly recommend reviewing The Central Dogma post before diving into this.

Two.  The protein we are going to make is called RED. 

Three.  To simplify purification, we’re going to attach another protein to RED called GST.

Four. If you don’t follow this step, don’t be dissuaded from reading Steps 2 or 3.  I’m going to try and make each step stand on its own.  Much like a TV show that has an overarching story through several episodes, each episode can also be watched independently.  I’m going to try and make each post in this series be its own story.  If you see how all the pieces fit together – awesome!  If don’t, just read each post for the story it tells.

                This first step is called Cloning/DNA Preparation.

BACKGROUND/EASY EXPLANATION

                Let’s begin with a bit of background about proteins.  The Central Dogma post explains that proteins are strings of amino acids.  We have twenty amino acids in our body and they can be hooked together in any order and to any length.  A protein is a unique sequence of these amino acids.  A protein can be as short as tens of amino acids or as long as hundreds.  The key to making a protein is knowing its exact amino acid sequence.  

                I’m sure most people realize that chemists hook together and/or manipulate molecules.  It would seem to an outside observer of science that chemists would have come up with an easy way to link amino acids together in any order they want both quickly and easily.  If we know the amino sequence, we can just have fancy science hook them together the way we want, yes?

                The answer is no.  Nothing is more equipped to make proteins than cells.  Cells have all the necessary machinery to create proteins quickly and efficiently.  What takes chemists hours to do, a cell can do in mere seconds.  For this reason, if scientists want a particular protein, then we ask cells to make it.  We ask kindly and, in particular, we ask E. coli bacteria* to do it.

                Why bacteria?  They grow quickly (they double their numbers in ~ 20 minutes), we know exactly what temperature (37°C or 98°F for E. coli) they like and we know exactly what they like to eat.  In short, bacteria are easy to grow in large numbers in a laboratory.

The Central Dogma post reminds us that DNA leads to RNA which leads to protein.  If we want bacteria to make our protein, then we need to provide it with the instructions on how to do it.  The instructions are, of course, the DNA that encodes the protein.  

                Bacteria have their own genome (their own DNA instructions) and we can’t fiddle with that without messing up the bacteria.  Lucky for us, bacteria also have very small pieces of DNA hanging around inside them that are separate from their huge genome.  These little pieces of DNA are called plasmids.  Messing with them usually doesn’t mess up the bacteria!  So, if we can put the DNA that encodes our protein inside a plasmid and put that plasmid inside a bacterium, then it will know exactly how to make our protein.

                This step is called Cloning/DNA preparation because it is the step where we prepare a plasmid that has the DNA that encodes are our protein’s amino acid sequence.  For the purposes of this example, our plasmid will encode the protein GST-RED.  If we look at our picture from Figure 42.1, the result of step 1 is a circular piece of DNA.  The DNA that encodes for GST-RED is colored red and the remaining plasmid DNA is black.  

                Once we make our plasmid, we put it inside bacteria and then grow a lot of bacteria that have that plasmid.   This is moving into Step 2, though!

MORE INFORMATION/ INTERMEDIATE AND DIFFICULT EXPLANATIONS

Where do we get DNA?  In the Extreme Solution post, I discussed a technique called PCR.  If we can get a small amount of DNA that encodes the protein we want, then we can make a lot of that piece of DNA with PCR.  We get the initial bit of DNA from whatever organism makes the protein to begin with.  For example, if the protein we want to make comes from humans, we get human DNA.  From here, we use PCR to amplify the bit we want.  We have to amplify it before we can put it inside the plasmid DNA.  If you'd like to know more about this part, just ask, but I'm going to leave it at this for now.

Where do you get plasmid DNA?  Companies like Invitrogen and NEB make plasmids that bacteria will readily take up.  It’s just up to the scientists to insert the DNA they want into the plasmid.

How do you get the plasmid DNA into the bacteria?  The process is called transformation.  We take a small amount of bacterial cells and add in a bit of plasmid DNA.  The bacterial cells have been treated such that their outsides are sticky.  The plasmid DNA sticks to the outside of the bacteria.  We then drop the tube of bacteria into water at 42°C.  The heat causes the bacteria to swell and open up.  The plasmids that were sticking to the outside can then get inside.  After a short period of time (45 seconds to 1.5 minutes), we place the tube of bacteria back on ice.  This closes up the bacteria again so the plasmid DNA cannot get out.  Ta-da!  We have plasmid DNA now inside bacteria.

More questions.  No, I have not addressed every point in this process.  You can ask how scientists insert the DNA they want into plasmids, how we know we’ve done it correctly, and how we ensure that the bacteria keep the plasmids inside them (they have been known to spit it out).  These are all valid questions and if you are interested, I’ll give you the answers!  Just ask!

* - the easiest organism to ask to make protein is bacteria and that is what I'll use as my example here.  We can also ask mammalian cells or insect cells to make protein, as well.  The steps are overall the same but more involved.  

REFERENCES

Me, myself, and I

               

               

               

From DNA to Protein, Introduction

                 Many of my previous posts have discussed work with individual proteins.  In the case of Diabetes mellitus, the importance of insulin was paramount.  In the Spanish Influenza series, I focused heavily on the protein hemaglutinin.  Thiaminase I took center stage in my mini-series on Australian vitamins while Taq polymerase was key to the polymerase chain reaction in An Extreme Solution.

                How do we know these things about individual proteins?  Well, they were isolated from the rest of cells and studied individually.  

                How do you go about isolating a one protein from the soup of proteins available within the cell?  Ah.  This is what my next few posts are going to focus on.  These steps are commonly referred to protein expression and purification.  What I plan to cover is the most straightforward way to isolate protein, but should not be considered the only way to do so or even to study individual proteins.  Scientists have all sorts of tricks in their bags!

                Protein expression and purification is broken into three steps (Figure 42.1):

Step One: Cloning/DNA preparation.  The necessary DNA is represented by the circle.

Step Two: Bacterial growth/expression.  The bacteria (gray boxes) with our protein of interest inside (red triangles) are shown.

Three: Protein Purification.  Eventually we just want our protein (red triangles) away from everything else.

                In Figure 42.1, I’ve given you a pictorial overview of where we are going.  It’s not supposed to make sense right now.  However, I hope at the end of this series, it will make a lot of sense!

                Several layers of understanding exist for explaining the above steps.  Obviously I understand down to the extreme nitty-gritty, which is probably way more than any of you would like to know.  It is also quite easy to get lost in the details and confuse everyone with what I’m discussing.  For these reasons, I’m planning on breaking each post into two parts: the first part will fall under the EASY category and discuss very plainly what is going on; the second part will be more INTERMEDIATE/DIFFICULT and will discuss the same ideas as the first part, but in more detail and with more scientific considerations in mind.  Whether you read one or both parts is completely up to you!  I also won’t dive all the way down to the all minutiae so ask any questions you wish!