Wednesday, August 28, 2013

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 –

Functions of proteins –

Standard amino acids –

Peptide bond –

Protein structure –

Non-standard amino acids -

Wednesday, August 21, 2013

Life is Elemental

Biology concepts – elements, biomolecules, biochemistry, trace elements, selenocysteine, stop codon

The blue whale is the often 30 m (100 ft) long and can reach a
mass of more than 175 tons (160,000 kg). As such, it is the
largest animal ever to grace the face of Earth. Yet, it owes it
shares its intricate biochemistry with even the smallest
organisms on the planet. The commonality is due to the
chemical elements that make up the biomolecules of all living
things. A few elements are used in most biological compounds,
and many elements are used in just a few. This is today’s story.
A blue whale is the largest animal on the face of the Earth - ever. You could swing a tennis racquet while standing inside a chamber of its heart (left). Built very differently, the watermeal plant is the size of a grain of salt. Comparing the two organisms at the macrolevel is like comparing lug nuts and twinkies, or pink and Darth Vader. But looks are often deceiving.

At the genetic level, about 50% of the genes from whales and watermeal are exactly the same, coding for the same structural proteins or enzymes. At a biochemical level there's even more similarity; even if the gene products are different most of the processes that huge whales and tiny flowering plants carry out are exactly the same.

They are so similar for one overarching reason, and that reason points out an amazing commonality. Both the world’s largest animal and the world’s smallest flower come from a common ancestor. It may have been many moons since their family had that argument at the summer picnic that drove them apart forever, but they are still related nonetheless.

And since they have a common ancestor, they are going to harbor many of the same traits as that ancestor – including the ways they carry out the reactions and functions in their cells. The totality of the molecules that are present in an organism and how they interact to perform different jobs is termed an organism’s biochemistry.
Biochemistry is the chemical reactions that take place in living
organisms, like glycolysis and the citric acid cycle shown above.
Though different organisms may have subtle differences in the
proteins or even eliminate some of the steps, the overall
pathways are conserved across all life on earth. Look at the
molecules, the elements are common in availability and
common to all life. This is evidence of evolution and why
biochemistry is shared so completely.

Biochemistry refers to how information flows through organisms via biochemical signaling and how chemical energy flows through cells via metabolism. All life on Earth uses basically the same biochemistry since we all came from a common ancestor – to the best of our knowledge.

Organisms on Earth have similar biochemistry in part because they use the same types of macromolecules. Life as we know it is based on the interactions (biochemistry) of lipids, carbohydrates, proteins and nucleic acids. Each of these macromolecules is amazing and contains many exceptions, so we will deal with each in next few posts.

Whales and watermeal, all life for that matter, is organic (Greek, pertaining to an organ) since their biochemistry is based on carbon, but there many exceptions to our important molecules being organic. What is the most abundant molecule in living things? Water. Is water organic? No.

What creates the electrochemical gradient that fires our neurons? Sodium, chloride, and potassium. Are they organic? No. So the next time someone makes a joke about being a carbon-based life form, you can say you are just partly organic, and then let them ponder whether you are some kind of cyborg.

So what do the macromolecules have in common that is related to the biochemistry of life? They are made up of the same chemical elements. In fact, most all biomolecules are made up of just five or fewer different elements; carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), and sulfur (S).

Carbon’s importance lies in its ability to bond to many different elements, and because it can accept electrons in a bond or donate electrons to a bond. Carbon can bond to four different elements at the same time. This increases the possibility of complexity and is one reason our molecules are based on carbon. The situations are similar for oxygen, sulfur, nitrogen, and phosphorous.

One of the lesser abundant elements is sulfur since it is used in proteins as a structural element mostly, although it shows up in bone and other skeletal materials as well. Still, the average adult male (80 kg/175 lb) contains about 160 grams of sulfur; this would be about a salt shaker’s worth.

Carbon is the basis of life on Earth because of its ability to form single,
double, and triple bonds, and because it can bond with so many
different elements. On the left is the top right corner of the periodic
table, showing carbon and silicon in the same column (family).
Elements in the same family have similar chemical properties, so
scientists believe that life on other planets could be based on silicon.
This is how we got the look and feel of the alien in the Sigorney Weaver
movies of the same name. But, silicon is more abundant than carbon,
so why don’t we look like the alien?
Only two of the twenty common amino acids that make up proteins contains sulfur (methionine and cysteine). But don't minimize its importance just because it is present in only two of the protein building blocks. The sulfurs in proteins often interact with one another, determining the protein's three-dimensional structure. And for proteins, 3-D structure is everything  - their function follows their form.

Sulfur is important in other ways as well. Some bacteria substitute sulfur (in the form of hydrogen sulfide) for water in the process of photosynthesis. Other bacteria and archaea use sulfur instead of oxygen as electron acceptor in cellular metabolism. This is one way organisms can be anaerobic (live without oxygen).

In a more bizarre example, sea squirts use sulfuric acid (H2SO4) in their stomachs instead of hydrochloric acid – just how they don’t digest themselves is a mystery. Just about every element has some off label uses; we could find weird uses for C, H, O, N, and P as well. Heck, nitric oxide (NO) works in systems as diverse as immune functions and vasodilation (think Viagra).

So these are the “elements of life” – right? Well, yes and no, you can’t survive without them, but you also can’t survive with only them. There are at least 24 different elements that are required for some forms of life. Two dozen exceptions to the elements of life rule – sounds like an area ripe for amazing stories.

Some of these exceptions are called trace elements, needed in only small quantities in various organisms. It may be difficult to define “trace” since some elements are needed in only small quantities in some organisms, but in great quantities (or not at all) in others. Take copper (Cu) for instance. Humans use it for some enzymatic reactions and need little, but mollusks use copper as the oxygen-carrying molecule in their blood (like we use iron).

Let’s start with a list is of the exceptions; a list will allow you to do some investigating on your own to see how they are used in biologic systems.

Aluminum (Al)        0.0735 g
Arsenic (As)              0.00408 g
Tyrian Purple, or royal purple, is a dye made from the bodies of several
mollusks from the eastern Mediterranean. The spiny dye snail (left) is
one such mollusk that produces the purple dye from its hypobrancial
mucus glands. The dye is based on a bromine-containing compound that
the snails use to protect their eggs from microbial predators (right) and
for hunting. The dye was prized because instead of fading with time and
sun exposure, it actually became brighter. Used as early as 1500 BCE by the
Phoenicians, Tyrian Purple was worth its weight in silver for two
thousand years.
Boron (B)                   0.0572 g
Bromine (Br)            0.237 g
Cadmium (Cd)          0.0572 g
Calcium (Ca)             1142.4 g
Chlorine (Cl)             98.06 g
Chromium (Cr)        0.00245 g
Cobalt (Co)                 0.00163 g
Copper (Cu)               0.0817 g
Fluorine (F)               3.023 g
Gold (Ag)                     0.00817 g
Iodine (I)                     0.0163 g
Iron (Fe)                      4.9 g
Magnesium (Mg)      22.06 g
Manganese (Mn)       0.0163 g
Molybdenum (Mo)   0.00812 g
Nickel (Ni)                   0.00817 g
Potassium (K)           163.44 g
Selenium (Se)             0.00408g
Silicon (Si)                   21.24 g
Sodium (Na)               114.4 g
Tin (Sn)                        0.0163 g
Tungsten (W)            no level given for humans  
Vanadium (V)            0.00245 g
Zinc (Zn)                      2.696 g

You can see that for each element I gave a mass in grams. This corresponds to the amount that can be found in an 80 kg (175 lb) human male. But don’t confuse the mass found with the mass needed.

Barium (Ba) isn’t used in any known biologic system, yet you have some in your body. It is the 14th most abundant element in the Earth’s crust, so it can enter the food chain via herbivores or decomposers and then find its way up to us. You probably have a couple hundredths of a gram in you right now.

Bromine (Br) is a crucial element for algae and other marine creatures, but as far as we know, mammals don’t need any. In fact, this brings up an interesting thing about chemistry. Chlorine is integral for human life, just about anything that requires an electrochemical gradient will use chlorine, to say nothing of stomach acid (HCl).

However, chlorine gas is a chemical weapon that will burn out your lungs (and did in WWI). Bromine gas is very similar to chlorine gas - so elements that are useful as dissolved solids can be lethal as gasses.

How about something supposedly inert, like gold (Ag)? We use it for jewelry because it is rare and supposedly it doesn’t cause allergy (wrong - see this previous post). But some bacteria have an enzyme for which gold is placed in the active center. Gold is rare, so why would it be used for crucial biology? Most elements in biology are more common.

Finally, we should describe a couple of the uses of non-standard elements:

Selenium in proteins is important for stopping damage from
oxygen, but in case you don’t think that is important enough,
how about insulin function. From the cartoon above, you can
see that selenoprotin function affects insulin responsive
elements (IRS) that in turn control DNA function, cell survival
(Akt), and carbohydrate management.
Selenium is a rare element, being only the 60th most common element in the Earth’s crust. Yet, without 0.00408 grams of selenium on board, a human is only so much worm food. Selenium is only essential for mammals and some higher plants, but it performs a unique role in those organisms.

In a few proteins, particularly glutathione peroxidase, selenium will take the place of sulfur in certain cysteine amino acids. Selenocysteine is an amazing exception because it is not coded for by the genetic code! Instead, the stop codon, UGA, (a three nucleotide run which calls for protein production to stop), is modified to become a selenocysteine-coding codon.

The selenocysteine amino acid changes the shape of the protein, and is found to be the active site for proteins such as glutathione peroxidase and glutathione S-transferase. These enzymes are crucial for cellular neutralization of reactive oxygen molecules that do damage by reacting with just about any other cellular biomolecule.

So selenocysteine is an endogenous biomolecule that is important for protecting our bodies – as important as the antibiotics we use from other organisms. But a 2013 study shows that some antibiotics (doxycycline, chloramphenicol, G418) actually interfere with the production of selenocysteine proteins by inhibiting the modification of the UGA codon. In many cases, the amino acid arginine is inserted instead of selenocysteine, reducing the functionality of the enzymes. Yet another reason to not overprescribe antibiotics.

One last exceptions - silicon is important for many grasses. Remember, this is silicon, the element; not silicone the polymer used in breast implants and caulk; and not silica, the mineral SiO2. Silicon is taken up by grasses of many types; crops, weeds, and water plants (although silicon in grasses may take the form of silica).  

Silicon (top) is an element that is used in many ways, including
in computer chips. Silica is a combination of silicon and oxygen
(middle) which is part of many products as well, including the
lightest material on Earth, aerogel, used in NASA projects.
Silicone is a rubbery material (bottom) that is used in caulks and
in many other things, including creepy movie prosthetics.
In some grasses, the inclusion of silicon makes them less likely to be victims of herbivory (being grazed on by herbivores). Herbivores avoid high silica-containing grasses because they aren’t digested well. A 2008 study showed that this reduced digestibility is related to silicon-mediated reduction in leaf breakdown through chewing and chemical digestion.

Another protective function of silicon in grasses was illustrated by a 2013 study. In halophytic (salt-loving) grasses that live on seashores, increased silicon uptake resulted in increased nutrient mineral uptake, and increased transpiration, the crucial process for water movement through the plant.

In addition, these plants have better salt tolerance in the presence of increased silicon, even though they already have specific mechanisms for reducing the damage that could be induced by such high salt concentrations. Silicon reduced the amount of sodium element found in the saltwater grasses. Pretty important for an element that is considered non-essential.

Next week, let’s start to look at the biomolecules made from C, H, O, N, P, and S. Proteins are macromolecules made up of amino acids, and amino acids are exceptional.

Tobe R, Naranjo-Suarez S, Everley RA, Carlson BA, Turanov AA, Tsuji PA, Yoo MH, Gygi SP, Gladyshev VN, & Hatfield DL (2013). High error rates in selenocysteine insertion in mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin. The Journal of biological chemistry, 288 (21), 14709-15 PMID: 23589299
Mateos-Naranjo E, Andrades-Moreno L, & Davy AJ (2013). Silicon alleviates deleterious effects of high salinity on the halophytic grass Spartina densiflora. Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale, 63, 115-21 PMID: 23257076

For more information or classroom activities, see:

Elements of life - 

Trace elements in diet –

Trace elements in plants –

What is biochemistry –

Sulfur –

Bromine –

Selenium/selenocysteine –

Silicon based life –