The Namesake (Chemistry, History)

Amedeo – masculine, Italian; Italian form of Amadeus

Pronunciation: ahm-a-DAY-o  or  ahm-a-DEE-o

Amadeus – masculine, late Roman: derived from Latin amare “to love” and Deus “God”

Pronunciation: ahm-a-DAY-us   or  ahm-a-DEE-us

(courtesy of www.behindthename.com)

                I chose the Italian chemist Lorenzo Romano Amedeo Carlo Avogadro di Quaregna e di Cerreto (who was also Count of Quaregna), more easily referred to as Amedeo Avogadro, for the namesake of this blog.  Holy long name.

Before we go any further, let’s look at some pictures of this gentleman (Figure 10.1).  He looks smart, huh?  I can’t believe he was put on a postage stamp!  How cool.

                Avogadro was born on August 9, 1776 (he just missed the Declaration of Independence!) to Count Filippo and Anna Vercellene in Turin, Italy.  In 1796, he received his law diploma and worked as a lawyer until the natural sciences started to seduce him.  He finally left law in 1804 after presenting two papers to the Academy of Sciences in Turin on the subjects of electricity and metallic salts.  After being appointed professor at Lyceum at Vercelli, Avogadro published two memoirs that would place him squarely in the history of chemistry.

                Linus Pauling (a tremendous scientist in his own right) feels that Avogadro “was a man with an intense curiosity about nature.  He believed that a scientist should try to understand the world, and should not be content … simply to describe the world.”  Referred to as a kind, affable, and sincere by Edgar Smith, Pauling also felt that Avogadro was modest about his work, but not to a fault.  Avogadro did push the scientific community to understand and adopt his conclusions about atomic and molecular structure.  Unfortunately, like many before him, his work wasn’t fully appreciated in his lifetime.  It was left to Stanislao Cannizzaro to take up Avogadro’s cause and impress the importance of his work upon the community.

                Avogadro, exactly one month shy of his 80^th birthday, died in 1856.  He continued as a professor until 1830 and studied science until the end of his life.  He was a private man and little is known about either his wife, reportedly Felicita Mazzé, or his six children.  Following his death, a bust of Avogadro was placed at the University of Turin and the Scuola Professionale at Biella.

                So what was his great contribution to chemistry?  He figured out molecular formulas.  For example, he figured out that hydrochloric acid was always one hydrogen atom bonded to one chlorine atom (written as HCl, see Salty Water post).    

How did he do this?  Let’s start with his hypothesis.

Avogadro’s hypothesis (as published in 1811): at the same temperature and pressure, equal volumes of different gases contain the same number of particles.

                I’m sure to most of you those words are just a mish-mosh of nonsense.  What he’s saying is that if you have a corked flask of 1 L that you completely fill with Gas A and, sitting next to it, you have a corked flask of the exact same size filled completely with Gas B, then the number of particles in each flask is exactly the same (Figure 10.2).  Particles can mean atoms or molecules*.  

                This was a bold assertion but immensely helpful to chemists performing experiments with little understanding of atoms or molecules.       

                Let’s now set the scene to describe what was known about atoms and molecules at this time to show how Avogadro’s hypothesis fit in.  

Scientists knew of atoms but had no way of knowing exactly what a compound looked like.  This means that they knew water was a molecule composed of hydrogen and oxygen, but exactly how was a little murky. 

John Dalton (important chemistry guy) was able to figure out that water (and other known compounds) were always being made with the same proportions.  By mass, water was always two parts hydrogen and one part oxygen.  But, did that mean that a water molecule was H₂O or H₄O₂ or H₆O₃ (or even higher)?  All of these are viable possibilities because they are all two parts hydrogen to one part oxygen.  Molecules were small so they could not be seen or directly measured.  A link between the unseen (atoms, molecules) and the seen (measurable volumes, masses, something!!) was desperately needed.

                Around the same time, a familiar gentleman by the name of Joseph Gay-Lussac (hello, Absolute Zero post!) was fooling around with volumes of gases.  For example, he knew that if he mixed 2 L of hydrogen with 1 L of oxygen, he’d get 2 L of water vapor.  Why?  No one was too sure.

                Enter Amedeo Avogadro and his hypothesis.  He figured out a way to link Dalton’s calculations with Gay-Lussac’s experiments and definitively decide a compound’s molecular make up. 

                I’m going to explain this with simplified examples instead of real atoms.  I will show you two fake Gay-Lussac experiments, the results, and what was known (Figure 10.3).  Then we’ll apply Avogadro’s hypothesis to it and show how that leads to molecular formulas.  The same logic was applied to real data to determine lots of molecular formulas.

Illustrative Experiment, Part 1

Gay Lussac: I mixed 1 L of Gas X with 1 L of Gas Y and got 1 L of Gas U.

Dalton: I know that Gas U is equal parts X and Y.

Avogadro: Equal volumes contain and equal number of particles.  1 L of Gas X and 1 L of Gas Y and 1 L of Gas U all contain the same number of particles.  For simplicity’s sake, let’s say 1 L = 100 particles.  100 particles of X + 100 particles of Y gave 100 particles of U.  The only way to get 100 particles of U is if one X and one Y come together.  That can happen 100 times.  Dalton says U is equal parts X and Y, so a molecule of U = XY.

Illustrative Experiment, Part 2

                Gay Lussac: I mixed 1 L of Gas X with 1 L of Gas Y and 

                                    got 0.5 L of Gas U

                Dalton: I know that Gas U is equal parts X and Y

                Avogadro: Equal volumes contain equal number of particles.  100 particles of X + 100 particles of Y gave only 50 particles of U.  How do you only get 50 particles of U?  Well… what if two Xs and two Ys came together to form one molecule?  If Gas U is really X₂Y₂, then you’d only be able to make 50 of them.  If you can only make 50 particles, then the volume you’d see if 0.5 L.

                See how Avogadro’s hypothesis became the link between the unseen and the measurable quantities?  His work allowed scientists to finally determine the molecular formulas of many known compounds.  Most introductory chemistry students know Avogadro in another way: Avogadro’s number.  Again, this conversion provides a link between a measurable quantity (a weighed out mass of element/compound) and the number of atoms or molecules actually lying there.  His work also lead to further development of the Ideal Gas Law, something I’ll cover in a different post.  

                So, why did I pick him as a namesake for my blog?  His contribution to chemistry was enormously important and laid the groundwork for understanding atoms, molecules, chemical reactions, and everything that followed.  The meaning of his name also intrigued me.  The longer one is a scientist and studies how the natural world works, the more awed one inevitably becomes.  We are all tied to this universe and its laws.  On some level, we all respect the science that governs our world.  One is free to interpret “God” however one wishes, but I don’t have a religious interpretation of it.  I define the word as “however this world came to be.”  I see it much more as a broad term to refer to things we don’t yet or may never understand.  Rest assured, though, that thousands of post docs and graduate students are out there desperate to try and add a little nugget to our understanding every day. 

* This assumption only works because the size of a gas is so large compared to the size of an atom or molecule.

Avogadro’s Number = 6.02 x 10²³ atoms or molecules per mole of substance.  I won’t define a mole right now, but suffice it to say a mole is analogous to a dozen.  If you have a dozen cookies, you have twelve cookies.  If you have a mole of water molecules, you’ve got 6.02 x10²³ molecules of water.  That’s a lot of water molecules.

References

Edgar Smith. “Amedeo Avogadro.” Nature (1911) 88 (2196), pgs 142 – 143.

Hinshelwood and Pauling. “Amedeo Avogadro.” Science (1956) 124, pgs 708 – 713.

Peterson. “Avogadro and His Work.” Science (1984) 226, pgs 432 – 433.

Zumdahl, Steven S. “Chemical Principles, 4^th Edition” (2002) Houghton Mifflin Company, Boston, MA.

               

               

Spanish Influenza, Part 3 (Biochemistry)

        This post will wrap up my short series on the Spanish Influenza (for now).  However, these three posts are not indicative of everything that is known about the crazy 1918 virus or even about its proteins.  A quick search on PubMed (www.pubmed.com) of “Spanish Influenza Pandemic” yields 285 results!  Annual flu viruses are always studied, in addition to the thousands of other viruses that exist in this world.  The field is vast!

                If you can recall back to my first post on influenza viruses (Spanish Influenza, Part 1), I said that viruses use stealth to obtain entrance to our cells.  A virus’s ability to get inside relies on the virus’s ability to bind the cell.  Proper binding, much like sliding a key inside a keyhole, unlocks the front door of our cell and ushers the virus inside.  Influenza viruses rely on their coat protein hemagglutinin (key) to bind a specific molecule (lock) that hangs off the outside of our cell.  The virus is looking for a sialic acid that can be presented one of two ways: α2,3 or α2,6.  You don’t need to understand exactly what that means, but you do need to understand that α2,3 is found in birds, while α2,6 is found in humans.

                Part 2 of our Spanish Influenza special explained where scientists were able to find RNA for the 1918 hemagglutinin protein.  Through a series of steps (that aren’t important for this post), scientists were able to translate the RNA into amino acids, fold up the protein properly, and look at it.  They studied the 1918 hemagglutinin from all angles and compared it to hemagglutinins from other influenza viruses. 

                From the post Protein Folding, I discussed the four levels that scientists use to describe protein structure.  The first movie (movie! - those Ph.D. skills are paying off!) below shows you one hemagglutinin molecule from the 1918 virus (Movie 9.1).  Secondary structure (alpha helices and beta sheets) can be easily seen.  Also, this movie is showing you the tertiary structure of the protein.  It answers questions like “How does it fold up in space?”, “Where are all those alpha helices and beta sheets?”, and “What about random regions with no secondary structure – where are they?”  The movie offers you the ability to see the protein from several angles.

                The fourth level of folding tells us how many individual molecules must come together to make a fully functional protein.  In the case of hemagglutinin, the answer is three.  Movie 9.2 shows you how three hemagglutinin molecules come together.  This is what the protein looks like when embedded in the coat of the influenza virus and is fully capable of binding a soon-to-be infected cell. 

                Let’s take a moment talk about what the trimer looks like.  The entire protein complex resembles a bouquet.  The long stem region (which runs ~ 130 Å) is highlighted in Figure 9.1.  Look carefully and you will see that each hemagglutinin molecule offers one long alpha helix to the stem and these three alpha helices all wind around each other forming what structural biologists call a coiled coil.  This is a common way for alpha helices to come together in protein structures.  The “flower” part of our bouquet is called the globular head region.  The stem region and the globular head are known as protein domains, which are parts of the protein that fold up to form a structure independent of the rest of the protein.  (I’ll go into more detail about domains in a different post.)

                An area of great interest is the place where sialic acid binds.  This falls within the globular head domain (Figure 9.1).  Before we look at it, I want to highlight two key points:

1.      
      1. A sialic acid linked to a galactose in an α2,3 way looks significantly different than a sialic acid linked in an α2,6 way. 

2         2. Humans have α2,6 linkages in their respiratory tract; birds have α2,3 linkages in their digestive tract.  In order for an avian virus to infect a human cell, it must gain the ability to bind α2,6 linkages.  This should correspond to a visible change in the protein at the site of sialic acid binding.

Okay – on to the area!!  What does it look like?  How does it compare to known avian hemagglutinins?

Figure 9.2 is directly from the paper and depicts the binding pockets for sialic acid.  The deeper brown represents atoms set further back from the atoms in lighter brown.  This is trying to give you a sense of depth to the pocket.  The top panel shows 1918 hemagglutinin while the bottom panel shows an avian hemagglutinin.  They look quite similar, yes.  But remember that the bottom hemagglutinin can only bind α2,3 linkages but the top one has gained the ability to bind α2,6.  Subtle differences must exist.

Check out the middle panel.  That is a hemagglutinin from swine that can bind α2,6 linkages.  The pocket is wide at the center.  Compare again the top and bottom hemagglutinin.  The top one is slightly wider than the bottom one.  This widening of the pocket must be responsible for its ability to bind α2,6 linkages.  It wouldn’t take much to widen this pocket – a simple amino acid change or two (which happens naturally) can widen it just enough to adapt the virus for human cell infection.

Of course, the widening of the pocket does not completely answer why 1918 influenza was so virulent.  Our immune system “remembers” different proteins from invaders and hemagglutinin is one of them.  However, look how little the pocket widened from an avian hemagglutinin and how dissimilar it looks to the swine hemagglutinin, which is fully adapted to binding α2,6 linkages.  This protein had barely changed from a bird virus and it is easy to conclude that humans had never seen anything like it before. 

 From year to year, hemagglutinins that can bind α2,6 linkages vary slightly or are placed with new neuraminidases (Spanish Influenza, Part 2) to make new viruses.  This is why we need a new flu vaccine each year – the viruses do change slightly.  However, our immune system has seen similar proteins and can still wage a small scale attack on these viruses.  In the case of 1918, this protein had never been seen before.  Our immune systems basically looked at it and said “What the hell is that?” while the virus promptly decimated our cells. 

Scientists continue to study this virus to obtain clues to why it was so virulent.  Widening of the pocket, never seen before hemagglutinin, different cleavage loops (something I didn’t talk about), etc. are all clues to what create an especially deadly virus.  If we know what the dangers are, then we can identify them before they become a widespread pandemic.  At least, that is the theory! 

Coiled coil: common structural motif in proteins where alpha helices wind around each other much like individual strands of rope wind around each other to make a stronger rope.

Domain: part of a protein that folds into its own structure and has its own function even in the absence of the remaining protein/amino acid sequence

References

Stevens et al. “Structure of the Uncleaved Human H1 Hemagglutinin from Extinct Influenza Virus.” (2004) Science 303, pgs 1866 – 1870.

PDB Code for 1918 hemagglutinin: 1RD8 (www.pdb.org)

Personal movies and figures were rendered in PyMOL.

               

Mishaps (Lab Life)

           I like to believe I’m laid back in lab with both myself and my students.  Just don’t blow anything up and it’ll be fine.  You make a mistake?  Okay.  You forget to add something?  That’s fine.  You have no idea what you’re doing?  Let’s explain it again.  Not a problem.  I aim to be a good scientist without being anal about the things that don’t really matter.  A manuscript or a publication worthy experiment?  Get your serious face on.  An initial attempt to see what the hell is going on with a particular protein?  Mess up all you want.  You’ll end up repeating yourself fourteen times before you get a real result, anyway.  Don’t sweat the small stuff.

Unfortunately, in my many years of lab work, I will admit to being a bit too laid back or casual about some situations.  Of course, I was taught proper protocol for working with dangerous things, but you get cocky after awhile.  You’ve done something so many times that you know exactly how it will go and become lax on certain safety precautions that you decide are “optional.”  

For example, I started scoffing at the idea of wearing full protective gloves with liquid nitrogen because I was working with such a small amount.  “What’s the big deal?” I asked.  Yeah, well, I learned when I accidently froze my two fingers in it.  Frozen solid.  I could tap them (tap, tap, tap) on the bench top.  They were stark white, wrinkled as after a long bath, and frighteningly numb.  

 I’ve decided to share with you two stories about how I learned not to do a lab-related activity.  They are meant to be funny (as is this whole post) because it all ended okay.  However, there are situations in labs where things end poorly.  I’ve seen pictures of liquid nitrogen tanks exploding or fires that decimated labs and killed employees.  These stories below are not like that.  They are meant to make you smile just as a friend telling you she tripped over the dog and landed on the sidewalk with a skinned a knee would have the group giggling.  More importantly, none of my stories below would have put anyone else in my laboratory in danger.  They were silly mistakes born of ego and arrogance, not ignorance or irresponsibility to others.  Sometimes we all forget what we’re doing and end up with a cautionary tale to share.  These are a few of mine.  (For extra entertainment value, I’ve named them like Friends episodes)

The one where I poured a neurotoxin on myself.

Scientists regularly use a chemical called acrylamide.  It comes to us as individual acrylamide molecules floating around in a solution with the consistency of water.  When we add two other chemicals to it (TEMED and ammonium persulfate), over the course of several minutes, all the acrylamide molecules bind to each other and the solution turns to Jell-o in our tubes.  This stuff is quite useful!  We use the jello acrylamide (called polyacrylamide) to do all sorts of experiments.

Polymerized acrylamide poses little danger, but monomeric acrylamide is a neurotoxin.  Inhalation or absorption through the skin can lead to adverse effects on your nervous system.  I, of course, was well aware of its characteristics on this fateful day.  

It was summer so I had on a t-shirt, but the lab was cold from air conditioning.  My lab coat was, obviously, hung over the back of my lab chair because, even when presented with chill, I do not think to put it on.  I am most unfortunate to suffer from runny noses when the temperature is below 72 degrees whether I’m wearing long sleeves or not, so I was sniffing a lot.  (Why buildings pump air conditioning in the summer is beyond me – most people are dressed for the balmy temperatures outside and not the arctic temperatures inside.  Crazy.)  Anyway, here’s what went down:

I had an uncapped tube of monomeric acrylamide in my left hand.  The post doc across the lab asked me a question and I began to think about my answer.  I realized that my nose was running (again!) so I moved to rub it with the back of my left hand.  The only way to do this is to (of course) turn my hand over.  This is all well and good unless your left hand is holding an uncapped tube.  The unpolymerized acrylamide went all over my right forearm.  

For one paralyzing second, I stood there unsure of what to do.  Then, I raced to the sink and flushed my arm with water.  I then proceeded to the bathroom where I scrubbed my arm raw with soap and water.  Finally, I admitted to my boss what I did and he sent me off the emergency room.  I had to bring the MSDS (material safety data sheet) for acrylamide and explain my stupidity.  Luckily, the percent acrylamide I poured on myself was very low (4%), I showed not even a minor red mark on my arm (aside from the effects of my incessant scrubbing) and everyone agreed that I would be completely fine, which seems to be true. 

I have never even come close to doing this again.

The one where I inhaled fire.

Every Monday, large plastic tubs of dry ice are delivered to my laboratory building.  Every molecule on this earth can be in one of three phases: solid, liquid or gas*.  Carbon dioxide (one carbon bound to two oxygens) is typically in gaseous form.  We breathe it out while plants suck it in.  (It’s a wonderful little circle we create.)  If you cool carbon dioxide down to a very low temperature, then it will form a solid.  This is analgous to cooling water down until it forms ice.

Now, let’s remember: Humans breathe carbon dioxide out.  We do not want to breathe it in because it will suffocate us.  In fact, our building has a rule that no dry ice is allowed on elevators due to the risk of elevator breakage, carbon dioxide take over, and scientist death.  (Not good for business.)

I use dry ice in my lab nearly every day.  It’s cold so I try not to touch it too much, but further respect for it ended right about there.  Until the day I went the tub and found only a small amount in the bottom, that is.  I sighed, grabbed the scoop, and plunged my face all the way to the bottom of the tub to get the last few nuggets.  This is when, for reasons still unclear to me, I took a deep breath.

Um.  There is only way to describe this feeling: FIRE.  Hot, shooting, suffocating, brain-numbing fire-y pain.  It raced down my throat and burned my lungs then shot up my nostrils, surrounded my brain and hugged tight.  I wish I was being dramatic.  

I pulled my head out, took several deep breaths and rubbed my head.  After staggering back to my lab, I asked my friend if he ever put his head to the bottom of the dry ice bin.  He looked at me very seriously and then said with a big smile,

“You took a breath, didn’t you?  Hurts like hell!”

I guess we all have this rite of passage.

Final Notes

Neurotoxins and dry ice should be treated with respect, as should most laboratory equipment. 

Acrylamide can come as a powder form that will leave particulate matter floating around in the air.  Inhalation of this powder is dangerous.  Most labs now won’t have powder and strictly order it in liquid form.  The percent acrylamide ordered is also around 30 – 40% so spillage is less likely to lead to harmful effects in scientists.  Accidents do happen so the field has built in safety precautions with this essential chemical. 

Also, dry ice can kill people.  I’ve heard anecdotal stories of scientists finding someone in the bin the following morning.  In truth, it’s hard to fall inside the tubs we have unless you climb inside (geez – why would you do that??), but stranger things have happened.  I found a paper from the Journal of Emergency Medicine that reports on the death of a scientist from dry ice left in a walk in freezer.  Simple safety precautions, like only using dry ice in well ventilated areas, are things that are taught very early on to younger scientists.

* Some will argue that a fourth phase can exist called "plasma."  It only seems to be present in certain places (like stars) so it's commonly ignored or mentioned in passing by biochemists.

References

Dunford et al. “Asphyxiation due to dry ice in a walk-in freezer.” The Journal of Emergency Medicine (2009) 36(4) pgs 353 – 359.

Me, myself and I.

Protein Folding (Biochemistry)

I apologize in the advance for the overuse of the words “protein,” “fold,” and “structure.”  There’s just only so many ways to say these things!  But, there are lots of colorful pictures in this post.

 

                The central dogma (see post of the same name) explains how we get from DNA to protein and tries to underscore the essentiality of proteins to our existence.  They are the work horses of cells and, without proteins, we would not be alive.  Proteins carry oxygen around our bloodstream (hemoglobin), they catalyze reactions that would not otherwise happen (enzymes), they protect our cells against damage (p53), etc.  The list is seriously endless.

                What do proteins look like?  So far, I’ve explained that proteins are strings of the 20 different amino acids.  What does that mean?  

Let’s first look at amino acids (Figure 7.1).  All amino acids are small molecules that have an amine group (where we get the “amino” part) on one end and a carboxylic acid group (where we get the “acid” part) on the other.  In between is one carbon that bears the “R” group.  Each amino acid has its own “R” group.  For example, if we are talking about glycine, the “R” group is a hydrogen.  If we are talking about cysteine, the “R” group is a sulfur bound to a hydrogen.  All amino acids share the same basic scaffold, but vary at the “R” position to give some variety (the spice of life, people).

                The ribosome is where all these amino acids are linked together.  Since each amino acid shares the same scaffold, they are all linked together in the exact same way: via a peptide bond (Figure 7.2).  The carboxylic acid of the first amino acid comes close to the amine group of the next amino acid and some magic happens: the N binds to the C to form the peptide bond, while the O plus two Hs (in the form of H₂O or water) leave the molecule.  

                The first amino acid in a protein (or polypeptide) still has a free amine and is called the N terminus of the protein.  The last amino acid has a free carboxyclic acid and is called the C terminus of the protein. 

                With the facts listed above and a known sequence of amino acids, you could draw an entire protein molecule.  (Yup – you could!)  Your gorgeous drawing would be called the primary structure of the protein.  

                I’m sure you realize, however, that proteins are not just long straight lines of amino acids floating around.  Think of a pipe cleaner – sure, it can be a long straight line, but it can also fold up on itself into any number of conformations.  Ah, the same is true of proteins!  The long string of amino acids will fold up to form a three dimensional structure and, once properly folded, it will become a fully active protein.  Scientists further breakdown protein structure into these three topics: secondary structure, tertiary structure and quaternary structure.

Secondary Structure: Because peptide bonds are just linking essentially the same molecule over and over again, this leads to some predictable ways that individual amino acids within the long string can interact with each other.  Sometimes they will all wrap around each other and form a helical shape, known as an alpha helix or sometimes they will all line up to make a flat sheet, known as a beta sheet (Figure 7.3).  Not all amino acids are involved in forming beta sheets or alpha helices.  Amino acids not involved in either are said to be in “random coil.”  These types of amino acid arrangements help form the three dimensional structure of a protein and are referred to by scientists as secondary structure. 

 Biochemists (like myself) can use algorithms to predict secondary structure from a protein’s primary sequence. That sounds fancy, but honestly, we just type in the primary sequence of the protein and a smart computer with a smarter programmer far away does some complicated calculations and emails us back the prediction in about two minutes.  The computer assigns each amino acid to alpha helix, beta sheet, or random coil.  Mind you, this is just a prediction.  To know for sure what is going on, we need to see the protein’s tertiary structure…

Tertiary Structure: This level explains how the rest of the amino acids are arranged in relation to and around the secondary structures.  Figure 7.4 shows some tertiary structures of proteins to give you a sense of how different proteins can look from each other and how secondary structure is incorporated into the tertiary structure.  Both the right and left slides are showing the same thing for each protein: the right side is highlighting the secondary structure (can you see the alpha helices and beta strands?) while the right is showing the exact same view but with all the atoms filled in (called space filling model).  Think of the difference between the left and right as a tree with no leaves (left) and the same tree full of leaves (right).

Quaternary Structure: For many proteins, one properly folded protein molecule is enough to fulfill the protein’s function.  However, other proteins require another protein to interact with before becoming fully active.  For example, hemoglobin is really comprised of four identical protein molecules (in both sequence and fold) that are all hanging out together.  Quaternary structure tells us how many protein molecules must come together to form the fully active protein.  If it is just one, then the quaternary structure is a monomer.  If it is four, then the quaternary structure is a tetramer.  (No, we are not limited to one or four – everything is possible!  I’ve heard of proteins with a quaternary structure that is dodecameric!)   

Scientists know the full structures (primary – quaternary) of many proteins, but there are thousands of others out there on which we still have little information.  For many of these, all we have is the primary sequence of amino acids and a guess at secondary structure from the available algorithms.  Part of understanding how a protein works is seeing what it looks like.  However, obtaining tertiary and detailed quaternary structures is an arduous amount of work that involves nuclear magnetic resonance imaging or X-ray crystallography (suffice it to say, neither technique yields an answer in a day – more like several months to several years).

Wouldn’t it be lovely if we could look at a primary sequence of a protein and know how it will fold up?  Of course!  But, unfortunately, we can’t predict much beyond secondary structure (YET!)  Several labs all over the world are trying to work out algorithms that will look at the primary sequence and spit back out an estimate at the tertiary structure.  So far, it’s still a work in progress, but advances are being made all the time!

Okay!  I think we are coming to the end of background information on proteins.  Excellent – this means I can discuss lots of literature for you!  The next Spanish Influenza post will look at the tertiary structure of hemagglutinin from 1918 and other influenza viruses (woo!).  I also have some posts ready on p53/cancer and multiple folded forms of proteins.  Ooooh…

Amine group: a nitrogen bound to three Hs (or R groups)

Carboxylic acid group: a carbon bound to both a double bonded oxygen and a single bonded OH or O^-

Peptide bond: the bond which holds two amino acids together

Polypeptide: synonym for protein

Primary structure: the sequence of amino acids within a protein from N terminus (beginning) to C terminus (end)

Secondary structure: structures that amino acids held together by peptide bonds tend to form – three types: alpha helix, beta strand/beta sheet and random coil

Tertiary structure: how all the amino acids within the protein fold up or are placed in three dimensions

Quaternary structure: how many of each protein molecule must come together before an active protein is achieved

Alpha helix: coils of amino acids, type of secondary structure

Beta strand / Beta sheet: flat string of amino acids (beta strand), two or more strands coming together create a beta sheet.

Random coil: amino acids not involved in alpha helices or beta strands/sheets that adopt no set conformation at the level of secondary structure

ADDED NOTE: I covered a little of protein folding on Dr. Amedeo in a post called Protein Knotting if you'd like to read a little more!

References

Alberts et al. “Molecular Biology of the Cell, 4^th Edition.”  Garland Science, New York, New York. (2002).

Bryson K, McGuffin LJ, Marsden RL, Ward JJ, Sodhi JS. & Jones DT. (2005) Protein structure prediction servers at University College London. Nucl. Acids Res. 33(Web Server issue):W36-38.

All protein structures came from the Protein Data Bank (www.pdb.org) and were rendered in PyMoL.

Tales from the Bench (Lab Life)

               8 times out of 10, the experiments a scientist does fail.  So much of science is trial and error.  This does not mean that we don’t know what we’re doing; rather it means that we have fifty hypotheses to follow up on and only one is correct.  The other 49 times, we’re wrong.  How fast we find the correct answer is influenced by our experiences, our breadth of literature knowledge, plain old luck and, most importantly, by the lab gods (they are finicky bunch).  

                Experiments fail for all kinds of reasons: wrong hypothesis, improper controls, improper interpretations of previous results, complete failure of the scientist to do the experiment correctly (sometimes you just add the wrong thing or accidently knock stuff over.  It happens.  There’s no shame in repeating an experiment because you’re an idiot.).  

However, the most egregious failures are when the lab gods decide to hate you.  It doesn’t happen often, but when it does, everything you do in lab that week will fail for reasons completely beyond your control.  Hypothesis correct, experiment planned well, you actually drink enough coffee to pay attention to your protocols, but the lab gods still decide that you aren’t worthy and they break your experiments in downright miraculous ways.  This happened to me during the week of March 1^st.  I won’t go into all the problems, but I will tell you about the last straw.  The failure that made me say, “Okay.  I get it.  I GET IT.  You hate me.”

Earlier in the week, I had discovered that a particular protein I was working with had a minor error in it.  Unfortunately, this meant that I had to repeat a bunch of experiments I had done before Christmas.  It happens.  I was angry for about ten minutes and then got over it.  I ran a very simple experiment to change the protein to what I needed (the protein was too long so I had to clip off a small part).  This is a very standard thing to do and I’ve done it approximately four hundred times during my career.

When I was done clipping my protein, the protein was very dilute.  For later experiments and long term storage of the protein, I needed to increase its concentration.  Figure 6.1 shows what a dilute and concentrated protein sample look like.  In short, I wanted many more protein molecules in the same volume.

This is a common problem and the company Millipore manufactures concentrators.  They are two tubes that stack inside each other (Figure 6.2).  You put your dilute protein in the top tube.  The base of the top tube has a filter.  This filter is full of very small holes that allow small things (such as water molecules) to pass through but larger molecules (such as my protein) cannot pass.  If you apply a force to the whole tube, the dilute protein sample will be forced downward towards the filter and what can fit through will flow into the bottom tube.  Whatever cannot pass through will remain in the top tube.  We apply a force to these tubes by spinning them at a high speed (centrifugation).  

Okay.  I’ve done this more times that I seriously can count.  My entire Ph.D. involved purifying proteins, concentrating proteins and performing experiments with them.  In fact, a tremendous amount of biochemistry involves this procedure.  I’ve used Millipore concentrators for seven years.  

On Friday, when the centrifuge had spun for 15 minutes, I went to pull out my concentrator and to check how things were going.  Imagine my surprise when I found my concentrator shattered (Figure 6.3).  The top tube had broken loose, spun to the bottom of the second tube, and cracked the entire unit.  My solution of protein was all over the bottom of the centrifuge.  My boss asked me if I could get it out anyway.  Um, that’s like trying to recover your spilled soup from a trashcan.  Gross.

In seven years, neither I nor anyone in my lab had ever seen a concentrator break so badly.  I did everything correctly with the clipping of the protein, purified it properly, spun the concentrator at the proper speed, but I still have to repeat the experiment because of something that no amount of ability could have avoided.  Freakish freakish accidents are just the most annoying of all.

P.S. – I pulled some more protein out the freezer on Monday, March 7^th, clipped it, and purified it.  This time it went perfectly.  Clearly, I needed a weekend break.

Centrifuge: an apparatus that spins rotors at high speeds and creates centrifugal force on the samples inside.

Rotor: the apparatus that holds sample tubes for high speed spins

References

Me, myself, and I.

If you’d like to see a real concentrator, courtesy of Millipore: http://www.millipore.com/catalogue/module/c7715

They have pretty pink caps.  They used to be blue – I have no idea why it changed!

NOTE: Originally, this post should have been #9, hence why the figures are actually labeled 9.1, 9.2 and 9.3  I moved it up in the order (to #6) and while I changed the numbering in the text, I forgot to change it in the figures.  Sorry about that. 

Spanish Influenza, Part 2 (Biochemistry)

                “And bingo.  Dino. DNA.,” proclaimed the DNA molecule, which was followed by some congratulatory music.  Ian Malcolm (Jeff Goldblum) leans forward in his chair with a large grin plastered on his face.  Alan Grant (Sam Neil) returns the smile and looks back to the screen.

                Yes, that is a scene from Jurassic Park the movie.  I’ve seen it a time or two.  I’ve also read the book a few times.  It’s really a very wonderful story, except for all those deaths and the grand ego of the initial idea holder, John Hammond.  Oh well.  The helicopter ride looked cool.

                I’m sure you will recall that scientists in Jurassic Park found dinosaur DNA in a complicated place: the blood held within the abdomen of a mosquito which had fed on a dinosaur, but then met a sticky end by becoming overcome with tree sap that eventually solidified into amber.  Whew.  That’s a lot of coincidences, if you ask me.

                Interestingly, scientists hunting for the 1918 influenza virus also relied on some coincidences and fortuitous timing to obtain their query.  Unfortunately, they sought the intact virus, which was unable to be obtained; they had to be content with discovering viral RNA.  As you will see, viral RNA contains a lot of useful information for rebuilding parts of this deadly virus.

                Viruses are stealth.  They are small, carry only necessary equipment with them, and blur the line defining what scientists classify as “alive.”  Figure 5.1 shows a generic virus.  While drawn much like a cartoon, the major points of the virus are well represented.  The blue and red “spokes” poking out are viral coat proteins, which enclose the inside of the virus, protect the genetic material, and are responsible for binding to cells then securing viral entrance.  For influenza, these coat proteins are known as hemagglutinin (discussed  in Spanish Influenza Part 1) and neuraminidase.  Coat proteins are also recognized and remembered by our immune systems in the form of antibodies.  Should your body become infected again with a virus it has already encountered, typically your immune system will remember and vanquish the invader swiftly.

                The inside of our cartoon shows lots of coils.  These coils represent the viral DNA.  Since we all now know about the central dogma (see previous post of the same name), we understand that DNA carries all the information for making proteins.  When a protein is needed, the DNA encoding this protein (called a gene) is copied into an RNA molecule then translated by a ribosome to make the protein.  

                A human cell’s DNA molecule encodes for thousands of proteins.  In contrast, a viral genome encodes for very few proteins.  For example, the human papillomavirus (HPV) genome encodes for seven proteins.  Seven!  How does a virus get away with this?

                If you read the post called Spanish Influenza, Part 1, you’ll remember that I said viruses want to break into our cells and steal our cellular machinery.  Human cells are rather self-sufficient: they have RNA bases floating around to be incorporated into new RNA molecules, they have amino acids ready to be strung together to make proteins, they have ribosomes waiting to read RNA molecules, etc.  Viruses do not have these things!  They carry a genome, which dictates how to make viral proteins, but they are completely unable to make these proteins on their own.  They must gain entrance to a cell and use the cell’s RNA bases, ribosomes, and amino acids to make their proteins.  Viruses are like bakers with no bakery.

                In essence, when we are infected by a virus, the virus breaks inside our cells and overtakes our protein-making machinery.  Instead of making cellular proteins, the infected cell is instead churning out viral proteins.  

                If our scientists could get their hands on some cells infected by the 1918 influenza virus, they would find a tremendous amount of RNA in the cell that is encoding for viral proteins.  RNA molecules encoding hemagglutinin and neuraminidase, among the other influenza proteins, could be sequenced and then translated.  From that information, they would have the amino acid sequence for the proteins and could begin to compare them to hemagglutinins and neuraminidases from other influenza viruses.  How do they compare?  Same amino acids?  Different?  By asking the correct questions, scientists can begin to tease out what made this virus so deadly and, possibly, preemptively identify similar characteristics in current influenza viruses.

                Several papers discuss the patients from whose tissue samples influenza RNA was able to be extracted.  I will summarize below, but suffice to say, the information came from very interesting places!  

-           - 21 year old male, admitted to the Fort Jackson, South Carolina army camp hospital on 9/20/1918.  He died six days later suffering from influenza, bacterial pneumonia, and cyanosis.  His body was autopsied and some tissue samples were formalin-fixed and embedded in paraffin.   

  

-          - 30 year old male, admitted to the Camp Upton, New York army camp hospital.  He died after three days of acute respiratory failure.  During autopsy, some tissue samples were formalin-fixed and embedded in paraffin.

This last place is the most interesting of all!

-          - Teller Mission (now called Brevig Mission) was ravaged by the 1918 influenza.  Located on the Seward Pennisula in Alaska, 85% of the adult population was killed (72 people) in five days.  A mass grave in the permafrost was used for all the dead.  In August 1997, several bodies were exhumed and lung tissue was biopsied for influenza RNA.  One Inuit woman (of unknown age) provided the best sample for extracting RNA.

From here, a known protocol was used to recover the RNA from fixed cells, amplify the results and obtain the sequences for both hemagglutinin and neuraminidase.  Several studies went on to compare the sequences with other known sequences, some also tried to rebuild an influenza 1918 virus and study its characteristics, and some actually studied what the individual proteins looked like in three dimensions.  This is where our next Spanish Influenza post will lead – we will look (with colorful pictures!!) at the three dimensional structure of 1918 hemagglutinin.

Gene: a stretch of DNA which encodes for a particular protein

Cyanosis: blueness of the skin due to lack of oxygen in the blood

Formalin-fixed: Fixation preserves tissue from decay or damage and allows for further investigation of the cells.

References

Crichton, Michael. “Jurassic Park.” (1990) The Random House Publishing Group, New York.

Spielberg, Steven. (1993) “Jurassic Park.”

http://www.cdc.gov/flu/images.htm

Reid et al. “Origin and evolution of the 1918 ‘Spanish’ influenza virus hemagglutinin gene.” (1999) PNAS 96, pgs 1651 – 1656.

Reid et al. “Characterization of the 1918 ‘Spanish’ Influenza virus neuraminidase gene.” (2000) PNAS 97(12) pgs 6785 – 6790.

The Central Dogma (Biochemistry)

DNA >  RNA > protein.

                The central dogma of molecular biology is nearly summed up in that small diagram.  (I’ll explain why I say “nearly” at the end of this post.)  

So, what in the world does it mean?

I’m going to use an analogy to describe a cell and the central dogma.  It’s useful to equate a cell’s job with that of a small business.  I’m going to use a bakery as my example (because Cake Boss is a great show.  I’m rather certain if I wasn’t scientist, I would have become a pastry chef) to explain why certain things and activities are placed where they are.  An overview of this analogy is given in Figure 4.1.

                Let’s start with a guided tour of a cell.  Figure 4.2 shows a representative cell with the essential parts to this post highlighted.  A cell is bounded by the plasma membrane – anything inside the plasma membrane is inside the cell.  

Let us think of a cell as a bakery.  A successful bakery can faithfully recreate their goods over time by following specific recipes.  This requires a steady flow of supplies into the bakery, proper placement of equipment, and protection of the recipes from destruction.  A cell operates quite similarly: its job is to create proteins (baked goods), which carry out almost all of the biological process that keep every living being in this dangerous world.  Most people probably never think about the thousands of proteins chugging away inside their cells that keep them breathing, running, working, thinking and, in short, living.

                The recipes for creating proteins are stored in the cell’s nucleus in the form of DNA.  The nucleus is a compartment, or organelle, within a cell that is surrounded by a protective membrane.  Just as the recipes are essential to a bakery’s survival, so are the directions for properly making each and every protein within a cell.  The DNA is so essential to cell survival that it has its own host of proteins that fawn all over it, uncoiling pieces that need to be accessed, rolling up other pieces that the cell is finished with, and repairing any damage that might occur to the DNA over time.  If you think of a cell as a kingdom, the DNA is king and the nucleus is its palace.

                Protein synthesis (or the baking of cookies, muffins, breads, etc.) occurs in the cytoplasm of the cell.  This makes sense – keep the recipes away from hot ovens or messy kitchens. The cytoplasm is anything outside of the nucleus but inside the plasma membrane.

                Can you see a problem?  DNA, which holds the recipes for making proteins, is highly protected in its nucleus.  Protein synthesis occurs in the cytoplasm, a completely different part of the cell.  How is the information passed?  How is the recipe written down?

                RNA.  When a protein needs to be made, the DNA is copied into RNA.  This is known as transcription.  RNA is free to leave the nucleus and enter the cytoplasm.  It carries all the directions with it to a small unit in the cytoplasm known as the ribosome.  The ribosome reads the RNA and creates a protein in a process known as translation.  In our analogy, the RNA is acting as a recipe copy (so as not to ruin the original) and the ribosomes are the bakery workers and equipment that turn ingredients into baked goodies.

           
                Information flows from DNA to RNA to protein.

                Let’s now switch and talk about the language of these molecules.  How is information conveyed?  How is DNA “copied” into RNA?  How does a ribosome “translate” an RNA molecule?  What are the ingredients used to make proteins?  An overview of cellular language is given in Figure 4.3.

                The languages of DNA and RNA are essentially the same.  Both molecules link together small units called bases.  DNA uses four bases: A, G, C, and T.  RNA also uses A, G, and C, but uses another base called U in place of T.  

                A molecule of DNA is a long string of As, Gs, Cs, and Ts.  Much like a spread out charm bracelet, DNA is a long backbone (bracelet) with individual bases (charms) coming off the backbone.  Because DNA is a double helix, it is has another backbone and another set of bases on its other side.  If we know what one side says, then we automatically know what the other side says because of base pairing.  A is always paired with T and G is always paired with C.  Figure 4.4 gives you a nice diagram of a DNA molecule.

                This base pairing is also what allows RNA to “copy” the DNA.  When a protein needs to be made, the DNA which contains the information for that protein is unwound and the second arm of the DNA is pulled away.  A molecule of RNA is then created by base pairing with the necessary region.  If the DNA says G, then the RNA gets a C.  If the DNA says A, then the RNA gets a U.  Once finished, the RNA molecule leaves the nucleus and the DNA molecule goes back together as a double helix.  This entire process is known as transcription.

                The language of proteins is different because instead of using bases, it uses amino acids.  Twenty amino acids can be strung together in various orders to make the thousands of proteins in our cells.  How do we get from bases to amino acids?

                The ribosome.  The ribosome is a complex and, quite frankly, fascinating little unit that grabs the RNA and threads it through.  Each stretch of three RNA bases stands for one particular amino acid.  The ribosome reads the first set of three bases and grabs the appropriate amino acid.  The ribosome then reads the next set of three RNA bases, grabs the appropriate amino acid and links it to the first amino acid.  Then the ribosome reads the next set of three, etc…  So on until the ribosome reads a set of three RNA bases that means “stop.”  At this point, the finished protein is released from the ribosome and the RNA is typically degraded.  This process is known as translation and an overview of this process is given in Figure 4.5.

                A set of three RNA bases is known as a codon.  Each amino acid has several different codons that encode for it.  For example, GUU and GUC both encode for the amino acid valine.  For this reason, you can’t look at an amino acid sequence and know exactly which set of three RNA bases told the ribosome to add that amino acid.  However, you can look at an RNA sequence and know exactly what the DNA sequence must have been because base pairing has no ambiguity.  For this reason, the central dogma is most accurately depicted as this:

DNA < > RNA > protein

                If you have the RNA sequence, then you know both the DNA sequence and the protein sequence.  However, if you have the protein sequence, you can’t be sure of the DNA sequence that dictated it.  Information flows from DNA/RNA to protein, but not in the opposite direction.

                This process of transmitting information has been used by every living creature that ever has been or ever will be on this planet. 

References

Alberts et al. “Molecular Biology of the Cell, 4^th Edition.”  Garland Science, New York, New York. (2002).