Download Tertiary Protein Structure

Fundamentals I: Hour 2
August 13, 2010
Dr. Delucas
I.
Scribe: Martika Skipworth
Proof: Terry Call
Tertiary Structure of Proteins
Tertiary Structure of Proteins [s1]–(quick review)
A. We are talking about how all the secondary structural elements interact and come together to
form a tertiary structure.
B. It is how the secondary elements folds over to form a tertiary structure.
C. The scope of tertiary structure is thus long-range because we are talking about from one side of
a protein to another. If something effects the protein here. It can cause a conformational
change on the other side. It is much bigger than just the length of a single alpha helix. It is many
alpha helices put together and sometimes mixed with beta sheets.
II.
Globular Protein Functions[s2]A. I talked about this in one on my other lectures.
B. There are many functions of proteins in tertiary structure or globular proteins.
C. There are ???? at the bottom of the list because there are many more functions. You
can go on and on.
III.
How Do Polypeptides Fold into Three-Dimensional Protein Structures?
a. How do these polypeptides fold? Well you are going to get lectures on folding so im not
going to go into the thermodynamics of it.
b. Entropy plays a major role- trying to hide the hydrophobic amino acids from the water
environment is very important. Ive said that over and over so you should know that.
Hopefully when you get the lecture on folding you will get a better understanding of
that.
D. Peptide segments between secondary structures tend to be short and direct. Secondary
within the tertiary structures interact through hydrogen bonds. Water tends to be
sterically forbidden in some areas because bonds are so tight.
IV. How Do Polypeptides Fold into Three-Dimensional Protein Structures?
a. Ive said this over and over. Hydrophobic groups tend to cluster together in the
folded interior of the protein.
E. Proteins tend to fold in order to make the most stable protein.
V. Tertiary Structure(several important principles) [S5]
a. In terms of globular proteins, they create the maximum number of internal bonds and
minimize solvent content especially with hydrophobic amino acids.
b. Fibrous proteins create intermolecular bonds. The strands interact and shield hydrophilic
bonds from aqueous environment.
VI.
VII.
VIII.
IX.
Fibrous Proteins[s6]
a. Characterized by association of helical chains which coil around each other forming
coiled coils.
b. There are some unique repeats like I mention for Alpha keratin heptad repeat.
c. Collagen unique repeat – amino acid that is derivative of the original twenty. You are
going to get this lecture later so im not going to talk much about this.
Structure of Collagen[s8]
a. This figure is a triple helical structure: It has 3 alpha chains each coiled in left-handed
sense( Minor helix).
b. 3 alpha chains coiled about each other in right-handed sense (Major helix).
H-bonds in the collagen fold[s9]
a. There is a hydrogen bonding pattern in collagen.
b. From Gly-N groups to C=O function of the preceding amino acid in neighboring chain. Im
not going to question you on this. You will have a full lecture on this. I guess that’s Dr.
Miller. Ill let him ask you those questions.
Globular Proteins[s10]
A. Ive said this over and over. Dr. Delucas has stressed point 1 and 2 in lecture. Make sure you
know these.
b. There is not a lot of empty space in proteins. The empty space that forms small cavities is not
really “Random coil”. They tend to have specified conformation. In crystal structures you can
only see things that have one conformation. Crystallization of protein reveals the detailed
structure. Why? Its not an alpha helix so it might be a loop that’s hydrogen bonded. Its
interacting with other parts of protein .They have a structure that unique to the protein.
c. Some proteins move. Some proteins open and close. If this is happening you can see the
crystalline structure. If its opening it so a substrate can come in. We usually Co crystallize the
substrate to the protein so that it clamps down and we can see it.
D. Various elements and domains of proteins move to different degrees: to put it in perspective
an example of a conformational change: image a large room (large protein) and a marble
attaches to the outside small protein)-> this causes a large conformational change. Its pretty
interesting to see this change. We can see where it ends and what it started like because we
can see the structure with and without these other structures bound. And then modelers can
tell us how it transitioned from one point to the other.
X. Most Globular Proteins Belong to One of Four Structural Classes[s11]:
a. Proteins can be classified according to the type and arrangement of secondary structure.
b. There are four classes:
1.Alpha proteins are made of only alpha helices. They have random loops and coils that bind
them.
2. Beta proteins are made up of nothing but Beta sheets.
3.Alpha and beta proteins have alpha helices and beta sheets that intermingle or interact.
4. Alpha +Beta proteins have separate Alpha helices and Beta sheets. There are thousands of
examples of each of these structures.
XII. The Serine Proteases[s12]:
a. Today in the crystal graphic data base we have over 55,000 protein structures. Most of them
aqueous proteins. Of those 55,000 only about 1000 over them are membrane proteins.
Some of them are derivative of others. We are hurting on when it comes to understanding
membrane proteins. Membrane proteins make up about 30% of the human genome.
b. So one class of Globular proteins are the Serine Proteases: I talked all bit about how we can
sequence other proteins to them.
c. They all have some common features
(1) All have to have a Ser in their active site where they do their biology.
(2) They all have to have a His Asp, and this is known as “the catalytic triad”. I will give you
the lecture on proteases in about two weeks.
(3) Through gene evolution only two of the three are available:Example of class look at
different proteases in different species some have two( Ser and Asp), but these seem to
be less efficient. It became more efficient to add the third amino acid (His).
XIII . Comparison of the amino sequence of chymotrypsinogen, trypsinogen, and elastase[s13.
a. This slide was hard for him to even see it. You have the catalytic triad in a sequence. These
things a far apart but when it all folds up. They are only a few angstroms apart. i). Part of the
protein has to sit by the triad to be clipped. The other amino acids in that area are conserved.
That true in everything we look at biologically. Ill show you a great example of that as we go
forward.
b. Structure of chymotrypsin: There is the active site region. There is catalytic triad region the His,
Asp, and Ser. So with a ribbon drawing like that you can kind of see that. If I showed you a space
filling model you would see that there is actually a little groove right there where the bond ends
up getting clipped.
XIV. Calcium Binding Motif (EF hand model)
a. Bob Kretsinger of University of Virginia discovered the EF hand model
b. We see the Ef hand model all the time in proteins.
c. In this kind of a structure that binds Calcium had now been found in over 3000 proteins in
our body.
d. He named it EF hand- When you have an alpha helix and you want to describe it to
somebody. You start from the N-terminus to the C- Terminus and you name them A, B, C, D,
and E. There was this helix and a loop of about 14 amino acids. And then there was F. He
name it that because it kind of looks like that.
e. Calcium sits in coordination with 8 oxygens. Loop has 14 amino acids.
f. There are many proteins that loop like this.
g. If you made a helical wheel-> loop of palm of the hand would be charged (polar).
h. 3 Calcium binding proteins always have an 1.Asp group at the beginning of the loop.2.
Glycine in position 7 or 8. He was kind of unsure. 3. Hydrophilic amino acid makes a
hydrophobic pocket with in hydrophilic area to interact with proteins. The is conservation
across species and time.
XV.
Globular Proteins[s19 s20]
a. this structure has alpha helices and beta sheet character twist. The alpha and beta
structure is intermingled( not separate)[s 19].
b. When making drugs, scientist usually target protein pockets( the drugs fit into the
pocket).This blocks the natural substrate from binding. This is structure base drug design(
inhibits enzyme). Water effects drugs binding to proteins active sites.
c. Old way Benzene and aliphatic group. This make us waste compounds and takes along
time. New way to make compound: Benzene is covalently attached to compound, add heat
, and they have a machine that sucks out whatever doesn’t bind. They make 10000
compounds in 3 day or so verses 1 in 2 months( old way). When making drugs, they find
which compound binds the best and test them all[s20].
d. This is whats done today for drug discovery. It is pretty exciting. A lot of people at
Universities discover drugs. At first we didn’t think we could do this because we didn’t
have the manpower or money.
XVI.
Waters on the Protein Surface Stabilize the Structure[s22]:
a. Surface structure of proteins always include water. There surround by shells of water. The first
layer is tight because of polar interactions and hydrogen bonding. There is also loosely water.
b. If we do a crystal structure, we can see the first shell of water and a little bit of the second shell.
c. I’ve said all this. I’ve talk about the helical wheel and the hydrophobic interaction being on the
inside of the structure.
d. A lot of time we can’t see the hydrogen in the molecule because we need crystals that are
about 1 angstrom in resolution. Scientists make up where they think the hydrogen is in
reference to the oxygen. This technique is pretty accurate way to determine where waters are
and how they are oriented.
XVII. Four helix bundle is common in domain structure proteins)[s23][s24]:
a. What you often see in tertiary structures is the secondary structure elements alpha helix
shielding the inner portion and exposing the outer portion to water.
b. Hydrophobic (green) and Hydrophilic (red).
c. [s24] Here are some real life examples of four helix bundles
d. They are not all parallel. Sometimes they have alittle bit of a twist.
XVIII: Many proteins are composed of several distinct domains[s26]:
a. Several domains interact. Loops can interact with protein or domain. They are ordered, with
hydrogen bonding, and they come together to make tertiary structure. Most of these a fairly
ordered with hydrogen bonding between them. A lot of times when we see multiple domains
Homo domains->gene duplication.
b. Different domains-> gene fusion which leads to new proteins. 1000 have been discovered and
there are many many more. We just don’t know how many yet.
XIX. Structure and Function are not Always linked [s27]
a. I think I gave an example of this yesterday. Thats all I mint to point out.
XX : Denaturation leads to Loss of protein structure and Function[s29]:
a.Heat protein to 50 -55C until hydrogen bonds are no longer strong enough to hold proteins.
Disulfide bonds may not be broken so you may have to add a reducing agent.
b. Don’t make a protein unless you know it is folded correctly. To do this add heat: if folded
properly ->protein will unravel at 50-55C. If it is not folded properly->protein will unravel are 3040C. If it is not folded correctly it will not be stable.
c. A differential scan calorimeter is used to detect when a protein starts to denature by scanning
the temperature of the solution .
XXI: Denaturation Leads to Loss of Protein Structure and Function[s30]:
a. Lysozyme causes the egg to denature when heated up.
b. It is easy to make proteins in eggs because eggs only have about 14 proteins in them so less
purification involved
XXII. Denaturation Leads to Loss of Protein Structure and Function[s31]
a.Figure on slide 31 You can look at the graph to see at what temperature the protein starts to
denature. Its in your book. Around 50 or 55 degrees Celcius is when a protein starts to denature.
XXIII. Denaturation Leads to Loss of Protein Structure and Function[s32]
a. Figure on slide 32 You can look at the graph to at what concentration of chemical the protein
starts to denature[s 32].
XXIV. Folding of Globular Proteins[s33]
a. How many times do I say this?? This is a very important concept. I didn’t realize that this was in
there so many times.
XXV. What is the Thermodynamic Driving Force for Folding of Globular Proteins[s34]?
a. Since you are going to get a lecture on this skip the folding slide.
XXVI: Marginal Stability of the Tertiary Structure makes Proteins Flexible[s35]:
a. If you look at all proteins and all interactions the have to be weak when it comes to biology.
b. Intermolecular interactions must be weak. We have to have an equilibrium.
c. Proteins have to be flexible to accommodate when something interacts. They cant be both weak
and strong. This would make the molecule unstable. Proteins have to be flexible. Small
conformational changes are weak doesn’t much change on molecule
XXVII: Motion is Important for Globular Proteins[s37].
a. Proteins vibrate very fast. The image would be blurred if it were played in real life at the original
speed
XXVIII: Layer Structures in Globular Proteins[s38]
a. The sheets are parallel and are coming toward you.
b. Some are formed in a circle or barrel.
c. This is an example of a proteins internal structure being hydrophilic Not hydrophobic. In the
ring, Beta strands have R-groups coming up. Underneath R-groups are going down. The inside is
flat. If water was added to the middle-> the middle of the circle would be hydrophobic
XXIX: Slide of Picture of Influenza[s 39}
a.
b.
c.
d.
e.
What the heck is that? This is the flu.
So influenza has two transmembrane proteins.
To bind to the cell, HA has a pocket that grabs a sugar
To get out of a cell, NA protein clips cyalic acid(the same sugar that lets it in)
We did the structure of this in our lab about 15 years ago. At the top of NA (active site), there
are 11 amino acids that have not changed for the virus(even for all the different strands like the
bird flu)[s39].
F. Wow if we can make a drug to block this, it would be useful. There was a company that opened up
because of this. If you want to read about it, its online.
XXX: Slide 40: Homo tetramer(space-filling model). There are four binding sites. The red part is the drug
in the binding pocket. There are 4 identical proteins.
A. No name for this slide [s41]
XXXI: Slide 43 Hemoglutanin has a coil coil arrangement of alpha helices. The figure is in the book. Skip
this slide.
I don’t know what slide this goes with.
a.In your book, they talk about alpha 1 Antitrypsin . “A tale of molecular mouse traps and folding
diseases.” There are many diseases today that are caused by misfolding of protein.
Alpha 1 Antitrypsin usually blocks Elastase.
*Lung Story: The Left lung is different from the right lung.
b. If you have an insult to the lungs, Neutrophils come the release Elastase. Elastase attack the foreign
foreign tissues but it doesn’t know good from bad. How do we stop it so it only does a little bit of
damage? Alpha 1 Antitrypsin with a big loop that moves. The next slide shows you what happens. It’s a
Methionine containing group that binds Elastase. The loop has a lot of flexibility.
c. In heavy smokers the mechanism doesn’t work properly. Elastase is not degraded properly.
d. I have spent the last 6 years working Cystic fibroses foundation trying to crystallize its regulator
protein. They know its only one defect that causes this disease. Many companies today have found a
drug that helps chloride transport. This drug gets 30% better lung function. The drugs going in reverse
the conformational change.
Diseases of Protein Folding[s52]
a. Read this slide for your own knowledge.