As Many Exceptions As Rules: So Many From So Few

Biology concepts – protein, amino acids, non-standard amino acids, peptide bond

Severe dietary protein deficiency leads to distinct

symptoms, and if not resolved, death. Called kwashiorkor

(Ghanan word meaning “disease from second born”),

the deficiency leads to changes in osmotic potential in

the bodies cells as compared to their blood.

Hypoalbuminemia (low levels of the blood protein

albumin) lead to fluid leaving the vessels and accumulating

in the abdomen, called ascites. This often occurs when infants

stop nursing (like when a second child is born); they take in

enough calories but not enough protein.

Heterotrophic organisms, including us humans, must consume protein in order to survive. Meat is a great source, by far the best protein source per unit mass and the best for obtaining necessary protein subunits (amino acids). If you look at complete protein sources compared to caloric intake, four of the top five foods are: turkey/chicken; fish; pork chops; and lean beef.

Tofu comes in sixth and soybeans are seventh. This is why humans have sharp canine teeth – we're meat eaters. You can live happily (well, somewhat happily) as a vegetarian; you just have to work much harder at it.

So why is protein so important? How about, because it is one of the four major biomolecules and without it you die a horrible death? Sounds like a good reason to me.

Proteins reside in every cell of every living organism, from prokaryotes to your favorite uncle. There isn’t a job in a cell that proteins don’t have their hands in; proteins even perform numerous tasks at the extracellular level. Heck, that spider web hanging from your dusty Stairmaster is made of protein!

From prokaryotes to spiny echidnas to rosebushes, let’s look where proteins are involved in life. Proteins provide the structure from which cells hold their shape and onto which they build a membrane. Proteins do the talking, providing chemical signals and ways to sense chemical signals.

Proteins do the dirty work; as enzymes they put molecules together, cut them apart, and change their parts around. And most times, they make these reactions happen faster than they would otherwise and without being used up in the process.

Enzymes are specific for a very few molecules (called

substrates). Enzymes have a particular shape, and this

allows the correct substrate to bind and be acted on;

called the lock and key system. Notice that the

enzyme itself is not altered by the reaction, so it can

work again on another substrate molecule. However

there are exceptions – suicide enzymes are inactivated

by their own action, so they only work once.

Proteins allow for movement, like the contractile proteins in your muscles or the proteins that make up flagella and cilia. Proteins even act as defenders of the cell, as antibodies and myriad other immune molecules.

A typical cell may contain 10 billion protein molecules. However, not every cell has the same proteins. Many proteins are necessary for every cell, but others have specialized functions needed in only some cells. The exception is unicellular organisms. Their one cell must be able to produce every kind of protein they might ever need.

Space is at a premium, so cells can’t waste room on proteins that aren’t needed right now. Therefore, making protein must be efficient, tightly regulated, and fast. Over 2000 new protein molecules are made every second in most cells, while some proteins exist only to destroy unneeded or old proteins.

Humans can make about 2 million different proteins, but we only have about 25,000 genes that code for them. We accomplish this by having some genes produce many different proteins, just by changing the parts of the gene used. These alternative splice variant proteins may have different functions even though they come from the same gene. For example, the cSlo gene is required for hearing, and each one of the 576 different splice variants is responsible for sensing a different frequency. Biology is just so dang efficient.

Now that you know how important proteins are, let’s find out what they are. Proteins are polymers (poly = many, and mer= subunit) made up of bonded amino acid mers. Proteins come in many sizes; the TRP-Cage protein of gila monster spit is a polymer of only 20 amino acids, while the titin protein of your connective tissue is over 38,000 amino acids long.

Maybe we'll dig into the degeneracy of the genetic code when we talk about nucleic acids, but for now let’s just accept that DNA triplets code for different amino acids, and the order of the codons determines the order in which amino acids are linked to form a specific protein. The order of the different amino acids is the key. Why? I’m glad you asked.

Amino acids (or aa’s) are all small molecules made up of carbon, hydrogen, oxygen, nitrogen, and sometimes sulfur – five of last week’s “elements of life.” It’s the arrangement of these elements that makes an amino acid. Refer to the picture below for a visual aid. The central carbon is bound to four other things (often called moieities). One is simply a hydrogen. Another is an amino group (contains the nitrogen). The third is a carboxylic acid group. Get it? amino acid.

While not the most exciting images, these cartoons should

help you understand the structure of the amino acid (left)

and the building of the proteins (right). Each amino acid has

the same structure, except for whatever the R group might

be. The amino end of amino acid 2 is joined to the carboxylic

acid of amino acid 1. The next peptide bond would be between

the carboxy end of amino acid 2 and the amino end of amino

acid 3. Notice how water is created each time a peptide bond

is made.

The fourth group is what makes each aa different. Called an R group, this side chain can be small or big, neutral or charged, and gives the aa its properties. The R stands for something, but that story is just too long.

In glycine, the R group is merely another H, but in tryptophan it contains complex rings. We have talked about how tryptophan is the least used amino acid; it is bulky and introduces big bends in the peptide. We’ll show that bends, kinks and other interactions between aa’s are important for the protein function.

Most organisms can make all the amino acids they need, but mammals are the exception. We have abandoned (genetically) pathways for making some aa’s, so we must get them from our diet. These are the essential amino acids, of which there are nine if you are healthy. Tryptophan must acquired by all animals – good thing plants still have the recipe.

Ribosomes (made of proteins and nucleic acids) link the individual aa’s together in the order demanded by DNA via the mRNA. The bond that connects them is called a peptide bond, and is a “dehydration” or “condensation” reaction.

Look at the amino acid picture again; the peptide bonding process kicks out water, ie. dehydration (de = lose, and hydro – water). Water forms from seemingly nowhere, like condensation on your mirror. See how fitting the names are?

When in a protein chain (also called a peptide), the order of aa’s is called the protein’s primary (1˚) structure. The primary structure in turn dictates the secondary (2˚) structure, which is a folding of small regions of the protein based on the interactions of the side chains of closely associated amino acids.

In turn, the folding of small regions brings together aa’s from farther apart, and they fold up based on their interactions. This is the tertiary (3˚) structure of the protein. If a protein needs more than one peptide chain to be functional, the shape that those different chains form when they interact is called the quaternary (4˚) structure.

These cartoons can help you picture how an individual amino

acid can affect the structure of an entire protein. In the

secondary structure cartoon, there are two basic forms that

the nearby amino acids can form, helices and sheets, other

parts will form no patterned form at all. The tertiary and

quaternary cartoons are for hemoglobin, showing how non-

amino acids may be involved (heme), and how the

individual peptides fit together.

The hemoglobin that carries oxygen in our red blood cells is made up of four protein subunits. Why is this important – because what the protein does in life is completely dependent on its three dimensional shape. Lots of aa’s means lots of potential shapes. This is in itself one of the greatest exceptions, since one of the basic tenets of biology is “form follows function.” But with proteins, function follows form.

For the greatest number of possible combinations and shapes, it’s lucky that DNA codes for 20 aa’s. Or are there more? Proteinogenic aa’s are those that can be added into a growing peptide chain, and there are actually 22 of them. The two exceptions are selenocysteine (like cysteine with selenium substituting for sulfur) and pyrrolysine (like lysine with a ring structure added to the end).

We talked last week about the functions of selenocysteine and how it can be incorporated into a peptide even though there isn’t a normal mRNA codon dedicated to it. Pyrrolysine is similar in that it becomes coded for after the modification of what is usually a stop codon, in this case UAG (a signal to add pyrrolysine is located after the UAG codon).

Pyrrolysine is used by methanogenic (methane producing) archaea and bacteria. It's important in the active site of the enzymes that actually produce the methane. New research is showing that more organisms than previously believed use pyrrolysine. A 2011 study identified more than 16 archaea and bacteria with pyrrolysine coding mRNA modifications, but it looks like there may be more.

While the mammalian titin protein is the largest protein

known (38,136 amino acids), there is a close second in a

bacterium called Chlorobium chlorochromatii CaD3. The

gene has been found for a protein of 36,000 amino acids,

but we don’t know yet of the protein is actually made. In

archaea, the halomucin protein from the square prokaryote

Haloquadratum walsbyi is 9,200 amino acids but is exported

to protect the organism from its extreme environment.

A 2013 study indicates that the typical modification of the mRNA that occurs 100 bp downstream of the UAG stop codon isn't even there in some pyrrolysine-coding genes. One hypothesis is that in genes without the modification, the UAG sometimes acts as a stop codon and sometimes incorporates a pyrrolysine. Therefore, there are truncated (prematurely stopped) and full-length versions of the protein in the cell, and the relative number of each can be affected by local conditions and stressors.

In this paper, the authors have developed a different predictor, which doesn’t rely solely on the presence of the modification. Using it, they have identified many new candidate genes in archaea and bacteria that could be using pyrrolysines. Here’s my question – all organisms use selenocysteine, but it seems only arachaea and a few bacteria use pyrrolysine. Why did it go away in higher organisms? Can it only be used for methane production? Please, no methane production jokes.

Pyrrolysine and selenocysteine are coded for by mRNA and are added to proteins, so we definitely have 22 aa’s, but could there be more? You betcha. There are over 300 non-standard amino acids, but that isn’t such a big deal. Remember the definition of amino acid; a central carbon with a hydrogen, a carboxylic acid, an amino group, and something else attached. It isn’t a wonder there are many of them.

Bacteria kill bacteria all the time. They make their own

antibiotics, called bacteriocins, by modifying short peptides

so that they interfere with cell wall synthesis in other strains.

To do this, they modify amino acids in peptides to non-standard

amino acids, including lanthionine and 2-aminoisobutyric acid.

Those that contain lanthionine are called lantibiotics and are

hot commodities right now.

A few non-standard aa’s can be found in proteins, like carboxyglutamate which allows for better binding of calcium, and hydroxyproline, crucial in connective tissue function. These are formed by modifying the amino acids already added to the growing peptide chain.

Other non-standard aa’s are produced as intermediates in other pathways and are not used in proteins. The list of them is great and their functions are even greater, but some act as neurotransmitters, others are important in vitamin synthesis, especially in plants. Still think life uses just 20 amino acids?

Next week we can finish up proteins. Life is very selective with the form of its amino acids – except when it isn’t.

Theil Have C, Zambach S, & Christiansen H (2013). Effects of using coding potential, sequence conservation and mRNA structure conservation for predicting pyrrolysine containing genes. BMC bioinformatics, 14 PMID: 23557142

Gaston MA, Jiang R, & Krzycki JA (2011). Functional context, biosynthesis, and genetic encoding of pyrrolysine. Current opinion in microbiology, 14 (3), 342-9 PMID: 21550296

For more information or classroom activities, see:

Dietary proteins –

http://www.fitday.com/fitness-articles/nutrition/vitamins-minerals/foods-rich-in-amino-acids-for-every-meal.html

http://www.wisegeek.com/what-is-dietary-protein.htm

http://www.webmd.com/fitness-exercise/guide/good-protein-sources

http://www.fitday.com/fitness-articles/fitness/16-surprising-sources-of-protein.html

http://www.care2.com/greenliving/vegetarian-protein-sources.html

http://www.healthaliciousness.com/articles/foods-highest-in-protein.php

http://www.hsph.harvard.edu/nutritionsource/protein/

Functions of proteins –

http://www.nature.com/scitable/topicpage/protein-function-14123348

http://learn.genetics.utah.edu/archive/protein/