Download Introduction to Proteins II

Fundamentals I: Hour 2
August 11, 2010
Dr. Delucas
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Proteins and Their Primary Structures
Scribe: Megan Guthman
Proof: Ashley Russell
Page 1 of 6
What Architectural Arrangements Characterize Protein Structure? [S22]
a. -strand depicted as flat arrow.
b. -helix depicted as barrel. Be familiar with these.
c. A -sheet, the way the atoms align, is shown here. (More in August 12 lecture)
The Sequence of Amino Acids in a Protein [S23]
a. The exact sequence of amino acids is unique for each protein.
Protein Structure [S24]
a. Is all the information necessary for a protein to fold just based on that primary sequence? The native
structure in how a protein folds – is it based completely on that primary sequence? The answer is yes.
b. Proven by an experiment Anfinsen and White did with bovine pancreatic ribonuclease.
i. Used urea, a heavy-duty detergent, and a reducing agent, -mercaptoethanol.
Bovine Pancreatic Ribonuclease A [S25]
a. If you look at the primary structure of the protein, you’ll see in yellow the disulfide bonds (cystiene-cystiene
bonds). They covalently link parts of the contiguous chain together. To do this experiment, they had to first
completely unfold the protein, then let it refold by itself and see if they could get an active protein. You can
test the activity of this because it’s an enzyme.
b. To break this apart, you have to add a reducing agent (-mercaptoethanol). The urea is added to keep it from
folding back until they want it to.
c. Surround the protein with a detergent and it keeps it open, also had to heat it to break disulfide bonds.
Example: When we want to look at the molecular weight of a protein, we do this with a different detergent but
we heat it up and if there are disulfide bonds we have to do the same thing.
d. [Back to the Anfinsen and White experiment] They did that, then started taking away reducing agent and urea
very slowly. The first time they dialyzed it all away – they put the protein in a bag, let all the small molecules
(urea and -mercaptoethanol) dialyze out into a buffer, changed the buffer and got rid of the rest of it, then
tested the activity and there was almost no activity. This little activity was better than when the protein was
totally denatured, so something happened. They heated it back up, put the reducing agent back in, and took
out just a little urea, then a little more, started going back and forth taking a little away each time. The activity
as they did this kept climbing
i. This experiment told them yes - everything that’s needed to tell how to fold in 3D is there. BUT there are
things like ribosomes, chaperones, proteins that help other proteins fold in our cells. If they’re not there, it’s
pretty much a random process. By doing it back and forth, eventually selected the most favorable
conformation energetically. Eventually got, not 100%, but a very active sample of the protein. Told them all
the information is there, but in a cell the protein probably needs help to fold.
ii. In a cell, as a protein is being made it’s being folded, it doesn’t fold at the end. That’s one thing they
couldn’t replicate here because it was one strand already made.
iii. Pretty much proved the primary sequence alone dictates that final conformation.
How are Proteins Isolated and Purified from cells? [S26]
a. Purifying proteins is important. If you want to biologically study a protein, separate it from everything else.
Has to be very pure.
b. A number of techniques to use. Similar to amino acid purification we already discussed.
c. Thousands of proteins in a cell can be separated. Affinity chromatography is one of the techniques used,
along with ion exchange chromatography, size exclusion chromatography.
d. One thing we can take advantage of is proteins tend to be least soluble when they’re neutral – an equal
number of (+) and (-) charges. If you know the sequence of a protein and you know the pH you’re working at,
and you know the pKa for all the amino acids, you can calculate the isoelectric point of a protein. If you know
the isoelectric point, you know that’s where it’s least soluble.
i. You could start adding salt to a mixture of protein. When you get a precipitate, you can then test if your
protein is in that precipitate. You can centrifuge it out, redissolve it, and see if your protein is still in there.
Everything that didn’t precipitate, you just separated it from about 20,000 proteins. A good first step.
e. [Insert “aldose reductase from placentas” tangent here] “Yes! It’s a placenta!” Moral of the story: times have
changed and so have research methods.
Figure: Solubility of Proteins at Different pH’s and Salt Concentrations [S27]
a. The ‘salting-out’ shown here for different salt concentrations. Shows if the isoelectric point is right here, you
see where the solubility, in terms of mg of protein, is going to be least at different concentrations.
b. Note in red type: salting in and salting out – what causes these to happen? It involves interactions of the ions
with the protein, and overcoming protein-protein interactions. Interactions of those ions to the point that it
involves water and ties up water molecules. When the salt concentration gets so high that it’s interacting with
the polar water molecules, it strips them from the protein itself. That water keeps the protein soluble, so at
Fundamentals I: Hour 2
Scribe: Megan Guthman
August 11, 2010
Proof: Ashley Russell
Dr. Delucas
Proteins and Their Primary Structures
Page 2 of 6
certain salt concentrations it’s happy. As you increase the salt and strip away the water, that protein is left
bare. It needs to shield itself, those polar molecules want something polar. So what do they do? They look for
the other thing that the salt isn’t interacting with as much – it’s another protein, the same protein. The protein
interacts with itself. It comes together, and as it does, it forms a big precipitate and falls out of solution.
c. How the salt ties up the water molecules to force the protein out of solution
VII. How is the Primary Structure of a Protein Determined? [S28]
a. The process is “divide and conquer.” You have a long chain and what we do is break it into smaller fragments
and then go analyze those fragments. If we have sequences that overlap in certain areas we can figure out
that one amino acid difference between one sequence and another.
b. Used to do this by hand chemically or with enzymes, today it’s done in an automated way: mass spec – done
by bombarding protein with gases that cause charges to be put on the protein. The mass of the protein when
you break it down is what we’re measuring. That is the most efficient way today. Older, classic methods in
book.
VIII. Frederick Sanger [S29]
a. In research, one of the first things we want to know is what’s on the end of the protein. Where does the
protein start and what is that amino acid? Sanger in 1953 sequenced two chains of insulin. He did that using
a process where he one at a time is clipping the amino acid, determining what that amino acid is.
IX. Chromatographic Methods are Used to Separate Amino Acids [S30]
a. If you want to separate amino acids, you can use HCl to digest them. That’ll break them up, it’ll quickly start
to degrade that protein by breaking it at the interface between each peptide bond. When you use acid to do
this, some amino acids are pretty labile, in HCL or any acid of that concentration, and you destroy the amino
acids. One of the first ones you end up destroying is tryptophan. Some of the other polar amino acids like
serine will eventually be degraded also. It is a way to break it all up but you end up getting false data because
you lose some of the information.
b. There are machines today that do this in an automated way. Use a variety of enzymes to break the protein up
into fragments and determine the exact number of amino acids in the protein.
X. The hormone insulin consists of… [S31]
a. Insulin protein that Sanger did the structure of. First had to break it into two chains, fragment those chains,
and sequence these one at a time. Did that the same way Anfinsen and White used to break disulfide bonds.
XI. Determining the Sequence – A Six-Step Process [S32]
a. If you look at a general method to do this – if you look at a polypeptide chain that’s more than one, like
insulin, [Step 1] first separate those two chains and purify them from each other. Those are different sizes so
you can purify them on some column that separates material based on size. [Step 2] Break disulfide bonds,
then determine with 2 different processes [Step 3]:
i. Edman degredation – identify the N-terminal amino acid, and if you want you can keep going, the next Nterminal one, and sequence the protein that way.
ii. Specific enzymes used to clip on the other end, the carboxy terminus. They’re listed here:
carboxypeptidase A, BC, and Y. Y is the most effective.
iii. These are two different ways to sequence: one from the N-terminus and one from the C-terminus.
b. [Step 4] Once you break these polypeptide chains apart, you then cleave them into smaller fragments. With
something large, you end up having a mess. Fragments of about 11 amino acids are easy to work with.
Sequence each fragment. [Step 5]
XII. Attaining Individual Chains [S33]
a. If the protein is a heteromultimer you can sometimes dissociate it by making the pH extremely high or low.
It’ll dissociate the proteins that are associating via ionic interactions or hydrophobic interactions. Detergent
like urea will do it. Guanidinium hydrochloride will also help break that multimer apart and help it stay as a
stretched out strand, lose all its conformational characteristics.
b. Reduce the disulfide bridges. (The protein would be denatured in this already.)
c. Purify the different peptide fragments, then follow the process that I had on the previous slide.
XIII. Fragmentation of the Chains [S34]
a. Can also use chemicals like cyanogen bromide to break proteins up.
b. There’s a number of enzymes that specifically cleave proteins at either the beginning or end of a specific
amino acid. By doing that, you know what amino acid Is just to the left of that enzyme. Helps you identify what
the amino acid is on the carboxy terminus or the n-terminus.
XIV.
Edman Degredation Reaction [S35]
a. This is the reaction, I’m not going to hold you to [memorizing it]. Both of these are nucleophilic attacks. You
end up creating a ring structure. You spit out the rest of the peptide chain. This derivative helps you identify
that n-terminal amino acid based on the R group it has on it.
Fundamentals I: Hour 2
Scribe: Megan Guthman
August 11, 2010
Proof: Ashley Russell
Dr. Delucas
Proteins and Their Primary Structures
Page 3 of 6
b. How would you identify that? All have characteristics in terms of reverse phase chromatography, where they’ll
come off. You could determine it just with a mass spec. You can do different chromatographic procedures.
These will all have a certain characteristic as to where they’ll come off. There’s a coulometric (yes, it’s a
word) ways you can do it also.
XV. Enzymatic Fragmentation [S36]
a. Trypin cleaves on the carboxyl side of a lysine or arginine. Since its on the carboxyl side where it cleaves,
you know that the last amino acid on a chain is one of these two amino acids. Chymotrypsin cleaves in a
different place.
b. They all act in different areas, and what allows that enzyme to act differently is a specificity pocket. If the
protein they’re cleaving has a positive charge in a long chain, it can fit into the pocket of the enzyme. When it
does, it can hold the chain there so the chemistry that occurs can break the peptide bond. If you have an
aromatic group, this enzyme has a fatter pocket that’s hydrophobic. It allows the aromatic chain to sit in there
and interact hydrophobically with the protein. In this case, the protein has a plus charge, and at the bottom of
the narrow enzyme pocket there’s a aspartic group with a negative charge. It’s all based on stereochemistry,
why that one cleaves right next to these, this one cleaves next to those.
XVI.
Trypsin is a Proteolytic Enzyme… [S37]
a. Shows where they’re cleaving and what you end up with. You can imagine if you cleave with trypsin, and go
back and cleave with chymotrypsin, you start to break these peptides down to almost single amino acids. You
do Edman degredation on those and identify that derivative with a chromatographic technique or with mass
spec and pretty soon you can put the puzzle together and figure out what the original sequence was.
b. Used to do this by hand, today it’s fully automated with a very small amount of protein.
XVII. Chemical Fragmentation [S38]
a. Shows the electronic interactions for a cyanogen bromide cleavage. Bromide is expelled, follow the chemistry
and see where the electrons are going. You end up with a cleaved product.
XVIII. Reconstructing the Sequence [S39]
a. This is reconstructing the cleavage. Taking the fragments and trying to figure out, based on each fragment
and doing Edman degredation from front to back on the smaller fragements, what the original structure was.
XIX.
Uses of Amino Acid Sequences [S40]
a. Sequence is important because it pretty much dictates how a protein is going to fold. The sequence, to some
extent, reflects function of that protein. If you look at proteins in chicken, dog, mice, bacteria, that have
sequences that are homologous, and they only have to be homologous 20%, the function is many times the
same. There are many cases where sequences are 80% homologous and the function’s not at all the same.
There are exceptions. But in general, if you have a lot of homology, usually the function in different species is
similar.
b. The sequence reveals the domain structure. Goal of NIH is to look at the ways proteins fold. Now we know
about half of them. We know if we see a certain sequence how it’s going to fold. You can predict domain
characteristics of the protein. Domains are parts of a protein that have different folding motifs.
c. Sequence can reveal differences in primary structure between different species or within same species.
i. Ortholog – similar in sequence between different species.
ii. Paralog – something like hemoglobin where we’ve had a gene duplication. Myoglobin only has 1 strand to
hold a heme group. Hemoglobin has an  and a  and there’s a gene duplication that occurred and there’s
a lot of homology between these two strands. That’s called a paralog. You can look for changes between
paralogs also.
d. Facilitate predictions of higher order structure – that’s what the modelers are trying to do from these
sequences.
e. Use the sequence to facilitate construction of probes for certain genes. Today there’s machines that quickly
probe the amino acid content looking for genes that may be combined to give you that protein. Many times its
portions of different genes that make one protein. We can use the sequence of the protein to predict the
different genes that combine to make that protein.
XX. Cytochrome C [S41]
XXI.
Chart [S42]
a. Within a species or between different species allows you to look at orthologous or paralogous interactions.
b. Cytochrome C has about 160 amino acids, the number of amino acid differences among cytochrome c
sequences of various organisms can be compared. The numbers bear a direct relationship to the degree of
relatedness between the organisms.
c. Each of these species has a cytochrome c of at least 104 resides. If you look at the chart, these are the
differences. It means that any species has more than half of its residues that are the same, they’re not
different. Human and chimp cytochrome C is identical. Even a plant, there’s not a lot of difference.
Fundamentals I: Hour 2
Scribe: Megan Guthman
August 11, 2010
Proof: Ashley Russell
Dr. Delucas
Proteins and Their Primary Structures
Page 4 of 6
d. Use this kind of information for many things – if you want to crystallize a human protein, look at other species
and how homologous they are to human proteins. With 30% homology between them, we can predict the
human structure from the other species’ structure with 3 Angstrom accuracy. Take diversity into account and
try to get crystals of any species’ protein. Instability of a protein might be because of a particular sequence of
amino acids, so you can compare the protein of choice to the protein in other species.
e. Can do the same thing when researching biological protein interactions. Often, a protein’s substrate binding
area is 100% the same across species. What varies is the amino acids that don’t matter. The part that’s
important for biology is almost always invariant.
XXII. Amino Acid Sequences of Hemoglobin and Myoglobin [S43]
a. Myoglobin v. hemoglobin and how there might have been a gene duplication from an ancestral gene.
XXIII. Figure: Evolutionary Tree [S44]
XXIV. This shows the time required to have a mutation. One mutation in a protein can totally affect its function. Ex:
sickle cell anemia. All it takes is a change in one amino acid. When you look at what it takes for these changes to
occur it’s pretty amazing. It’s rare to have a mutation and when you do it can be devastating, it can be what causes a
cancer.
XXV. The Unit Evolutionary Period [S45]
XXVI. Apparently Different Proteins May Share a Common Ancestry [S46]
a. Two proteins: lysozyme, and -lactalbumin. Lysozyme breaks down bacterial cell walls. -lactalbumin makes
lactose in mammary glands. Two different functions, yet, if you look at those proteins, they have sequence
homology and also look almost identical.
XXVII. Structures of Lysozyme and -lactalbumin [S47]
a. Crystal structures are extremely similar. Completely different functions. A case of functional diversity through
evolution. Lots of explanations for why this may occur, and you see it quite a bit as you look at structures
between species. Motifs can be identical but the two functions are very disparate.
XXVIII. Protein Purification and Characterization Techniques [S48]
a. Ways to purify proteins (addendum after chapter 5 in book).
b. They talk about dialyzing a protein, using affinity chromatography, size exclusion, ion exchange, hydrophobic
interaction, ultracentrifugation  all types of liquid chromatography.
c. Liquid chromatography – first one that came out is High-Performance Liquid Chromatography. Same kind of
things as above but on a much smaller scale – tiny columns under high pressure. Much better separation of
material than using packing material much larger in scale. Intermediate between this and the old fashioned
way doing it in columns that were very large.
d. Three techniques to characterize a protein after you purify it
XXIX. Dialysis [S49]
a. Can take a protein of molecular weight of 100,000 daltons and put it in a dialysis bag where the holes
between the bag are big enough to let anything 50,000 daltons get through. If the protein is in the bag and the
buffer is outside, and there’s some salt in there, it’s going to dialyze out.
b. If there’s smaller proteins than 50,000 daltons, they’re going to come out to some extent. They don’t always
come out completely but you get rid of a lot of it just with dialysis. Its one way to partially purify a protein.
Also, put protein in dialysis so its equilibrated in a solution with the right pH and salt concentration for the first
column you’re about to put the protein on. That’s what we usually use it for.
XXX. Ion-Exchange and Affinity Chromatography [S50]
a. Ion exchange chromatography is just what we talked about for amino acids, it’s no different. Pack a long
column with particles that have (+) charges and sometimes a combo of (+) and (–). Protein flows over this
and sticks to it. To knock the protein off you can either change the pH or increase the salt concentration. All of
those things change charge characteristics so they compete with the charges on the bound particles and they
knock your protein down the column.
b. When it comes off with protein, as long as your protein has one of the aromatic amino acids in it, you can
monitor it right at 280 nm, if it doesn’t have that you can monitor it closer to the peptide bond around 214 nm.
XXXI. Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of Charge [S51]
a. Some of the ion exchange materials. You’ll see (+) charges, (-) charges on these just like before.
XXXII. Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of Charge [S52]
a. Can use this for amino acids. Separating amino acids based on their charges and polarity.
XXXIII. Gel Filtration Chromatography Can Be Used to Separate Molecules on the Basis of Size [S53]
a. Another way we can separate proteins is based on how big it is.
b. Imagine a globular protein, and another globular protein that’s twice its size. You have particles that have
holes in it that the big one can fit through but it struggles. It takes it longer because it’s bumping into particles.
Those little particles there have interstices, holes and pockets in them. The small things get stuck in the little
holes. The big thing can’t so it slides around between the balls and the big particles come out first. The
Fundamentals I: Hour 2
Scribe: Megan Guthman
August 11, 2010
Proof: Ashley Russell
Dr. Delucas
Proteins and Their Primary Structures
Page 5 of 6
smaller ones come out second. You separate proteins based on how big they are. These are proteins in their
native state, not denatured, in their globular conformation. This is a very important technique that is used in
most labs today.
XXXIV. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) [S54]
a. If you take a protein and separate it by charge and it’s still not pure, you have 20 proteins left in there, now
you put it over a size exclusion column. You think it’s pretty pure, one way to test this is to characterize how
pure it is via a gel. We use a polyacrylamide to surround the protein in a detergent. What we want to do is
take that globular protein and find out if it’s pure.
b. To do that we open it up completely by putting detergent around it. If it has disulfide bonds we heat it and put
mercaptoethanol in there to break those bonds. Now a it’s linear strand surrounded by an equal charge all the
way around. All proteins in there have the same surrounding of charge. All we’re looking at now is the size of
the protein.
c. Now run it through a gel that, just like the size exclusion column, filters based on size. But now it’s not a
globular protein, it’s stretched out to be linear. When we do that, it gives very fine separation, sometimes
between proteins that are only 500-1,000 daltons in difference in molecular weight.
XXXV. Electrophoresis (SDS-PAGE) [S55]
a. This is what it looks like. Fill the wells with 10 micrograms of protein, run an electrical charge through here.
b. There are strands of polyacrylamide, it’s a polymer and the particles that are flying through here…the small
ones fly through first and the big ones get stuck. (Opposite compared to chromatography.)
c. Then we run standard proteins – we’ve denatured them and we know where they run. So we know the
molecular weight of the standard and it’s also run in one of these wells. If your protein travels to a certain
point, and there’s a standard line at that same point that is known to be 100,000 daltons, you know that’s the
molecular weight and that’s your protein.
d. To confirm that’s your protein, then run a similar thing, but instead of staining it with something that stains for
any protein, we stain it with an antibody that is specific for your protein. That’s called a western gel. When we
do that, the only things that shows up is your protein. If it’s the only band, we know it’s pretty pure.
XXXVI. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) [S56]
a. We can take proteins of different molecular weights and get a standard curve. As the molecular weight goes
up, it takes longer to move through the gel. You get a straight line curve to reflect the size of the proteins.
XXXVII. Two Dimensional Gel Electrophoresis [S57]
a. We can also take protein and do a 2D electrophoretic separation. This is nice because it can be used with a
sample that’s not as pure.
i. First, run isoelectric focusing. Isoelectric is where you have neutral charge. In this case, what happens is
you have a gel and you have analytes in there (we won’t go into that). Protein runs with a charge through
that gel until it gets to its isoelectric point, then it doesn’t move anymore. So you separate proteins based
on their neutral point, and hopefully you’ll see a bunch of different bands like the figure shows.
ii. Then take the gel and cut it out and lay it on top of the regular electrophoresis gel that separates based on
size of the protein. This would have SDS in it, etc. You now electrophorese in that direction, so it coud be
there were 10 proteins that had an isoelectric point of 4.5, now when you run this way you’d see 10 bands if
they have different sizes. It’s a 2D way to try to get lots of proteins separated and see what you have.
b. You can get fooled based on just size or just isoelectric point. There’s many proteins that have an isoelectric
point of 5.4. Having 2 different ways of looking at this to characterize it allows you to really see if you have
very pure material.
c. Today in research, we run SDS-electrophoresis, we cut the band out, and then we run mass spec. From the
mass spec, if there were 7 proteins in there, it identifies all of them based on the masses, fragments.
Generally, 2D Gel Electrophoresis is not used anymore.
XXXVIII.
Affinity Chromatography [S58]
a. Affinity chromatography is, in Dr. Delucas’ opinion, the most powerful way to purify a protein. You can take an
antibody, bind it to the beads in the column, and now your antibody is sticking out all over the beads in the
column. What’s your antibody going to bind to? Just your protein. Then you put your protein over that column,
and it should select out your protein and nothing else. This is a wonderful technique.
b. We don’t usually do it with an antibody because it’s expensive to make enough of it and do it routinely. There
are other things we can do, thanks to molecular biology. We can take a protein and when we go to express it,
put 6 histidines on the N-terminus. Histidines love to stick to metal. So what we do is put a column together
with metal like cobalt. Then we run the protein over, it sticks right to the top of the column where the cobalt is
in a nice flat bland. Then we wash the heck out of the column. Odds are, most proteins won’t have 6 histidine
residues, sometimes they may have 3 and you get some sticking. You get rid of 98% of your other proteins.
Now, you take something that competes with the histidine for that nickel group, imidazole is typically used
because it binds to nickel. When it does, it knocks your protein down the column. You get a peak coming off
Fundamentals I: Hour 2
Scribe: Megan Guthman
August 11, 2010
Proof: Ashley Russell
Dr. Delucas
Proteins and Their Primary Structures
Page 6 of 6
that’s hopefully just your protein. That’s the theoretical answer. In reality, it’s never just your protein but
sometimes we’ve narrowed it down from a million proteins to 20. Now we take that and go over a size
exclusion column, and then we’re pure.
XXXIX. How are Proteins Isolated and Purified from Cells? [S59]
a. This table shows as you start with a crude extract, the way to look at how good you’re doing is the specific
activity column. You can see that form step one, to the last step you went up 1000-fold in purity. That’s what
you want. The other thing is you don’t want to have to do too many steps to purify.
b. We always look for the specific activity of that enzyme to tell how pure a protein is.
XL. For Purification of Labile Proteins Less is More! [S60]
a. Take home lesson: when purifying proteins, especially labile proteins and membrane proteins: less is more.
Do as few steps as you can. Put a 10 histidine tag on, do affinity step, follow it with size exclusion, then run a
gel to see if you’re there. Start with something that’s pretty much going to get you pure then go one step
further.
XLI.
Purification of Cystic Fibrosis Transmembrane Regulator Protein (CFTR) [S61]
a. Real gels for cystic fibrosis protein. On the right is a silver stain. Looks great now, but in another day those
bands will break down and create more fragments.
XLII.
Purification of Epithelial Sodium Channel (ENaC) [S62]
a. When you run columns, you see peaks like this. This is the sodium channel.
[End 49:18 mins]