BIOCHEM DISCUSSION 2
So now we are going to talk about protein three-dimensional structure.
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Biochemistry: A Short Course Fourth Edition
CHAPTER 4 Protein Three- Dimensional Structure
Tymoczko • Berg • Gatto • Stryer
© 2019 Macmillan Learning
There are four different levels of protein three-dimensional structure, which we are going to talk about individually in this chapter, that is primary, secondary, tertiary and quaternary, for levels 1, 2, 3 and 4. Primary structure is just a linear arrangement of the amino acids. The secondary structure is some shapes that these amino acids can fold into on a local level. The tertiary structure is a way that all of these local shapes bundle together to form sort of globular proteins. The quaternary structure is how multiple proteins can come together and form a complex.
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Chapter 4: Outline
4.1 Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains
4.2 Secondary Structure: Polypeptide Chains Can Fold into Regular Structures
4.3 Tertiary Structure: Water-Soluble Proteins Fold into Compact Structures
4.4 Quaternary Structure: Multiple Polypeptide Chains Can Assemble into a Single Protein
4.5 The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure
So at the end of this chapter you will be able to compare and contrast the different levels of protein structure and how they relate to one another - so keep that in mind as we are going through this.
Now the primary structure is just the list of amino acids and the amino acids are linked together with what is called a peptide bond. In this protein, each amino acid is also called a residue (just another word).
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Section 4.1 Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form
Polypeptide Chains
Learning objective 2: Compare and contrast the different levels of protein structure and how they relate to one another.
• Polypeptides consist of amino acids linked by a peptide bond.
• Each amino acid in a protein is called a residue.
Figure 4.1 Peptide-bond formation. The linking of two amino acids is accompanied by the loss of a molecule of water.
A peptide bond is formed when two amino acids come together. A lone pair of electrons on the amino end (the nitrogen of one amino acid) attack the carboxylic carbon of the other amino acid and displace the oxygen creating water and a bond between the carbon and the nitrogen. The actual mechanism of this reaction is a little bit more complex but we will discuss it later in the course.
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Diagram of Peptide-Bond Formation
Now because each amino acid has an amino terminus and a carboxy terminus, therefore any polypeptide chain will also have an amino terminus and a carboxy terminus which means that they have directionality. And, when we write them we always write the amino terminus first. That is the way they get synthesized so that is how we will represent them. You can remember that very easily by AMINO-ACID, starts with an amino, ends with acid (the carboxy terminus).
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Polypeptide Chains Have Directionality
• A polypeptide chain has directionality. The amino terminal end is taken as the beginning of the polypeptide chain.
• The carboxyl terminal end is the end of the polypeptide chain.
• The primary structure is always written from the amino terminal to the carboxyl terminal, or left to right.
• A good way to remember this is AMINO-ACID – it starts at the amino terminus, and ends at the acidic (carboxy) terminus.
Figure 4.2 Amino acid sequences have direction. This illustration of the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL) shows the sequence from the amino terminus to the carboxyl terminus. This pentapeptide, Leu-enkephalin, is an opioid peptide that modulates the perception of pain.
This is an illustration of a pentapeptide, which is 5 amino acids attached in sequence. This particular pentapeptide is called Leu-enkephalin which is an opioid that modulates the perception of pain. There is a space filling model of this pentapeptide above and it is laid out in the same orientation as the drawing but in actual reality the molecule would be folded up quite a bit.
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http://molecules.gnu- darwin.org/html/00175001_00200000/183330/58822 -25-6-balls.png
So you can see on the bottom that the polypeptides consist of a backbone which is N- C-C-N-C-C, just on and on until the end, and then the side chains stick off of the side. The backbone has hydrogen bonding potential, because you can see the double bonded oxygens sticking off of the carbons and the hydrogens sticking off of the nitrogens along the backbone can hydrogen bond with each other - and that is extremely important for the formation of protein structures. There is a wide variety of proteins but the majority of them are between 50-2,000 amino acids long.
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Backbones of Polypeptide Chains • The polypeptide chain consists of a repeating part
called the main chain or backbone and a variable part consisting of the distinctive amino acid side chains.
• The backbone has hydrogen-bonding potential because of the carbonyl groups and hydrogen atoms that are bonded to the nitrogen of the amine group.
• Most proteins consist of 50 to 2000 amino acids.
Now something that may not be immediately apparent from looking at the previous diagrams is that there is a partial double-bond structure of the peptide bond and that means that it is in resonance with the double bonded oxygen. Because of this double bond, it restricts rotation around the peptide bond, keeping this peptide bond more or less planar {see next slide}.
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Polypeptide Chains Are Flexible Yet Conformationally Restricted (1/3)
• The peptide bond has partial double-bond character because of resonance; thus, rotation about the bond is restricted.
• The peptide bond is uncharged.
Figure 4.6 Peptide bonds are planar. In a pair of linked amino acids, six atoms (Cα, C, O, N, H, and Cα) lie in a plane. Side chains are shown as green balls.
And here you can see a representation of the planar nature of the peptide bond. You will have to imagine the double bond between the carbon and the nitrogen in the center.
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Model of Planar Peptide Bonds
The peptide bond is essentially planar. Six atoms (Cα, C, O, N, H, and Cα) lie in a plane.
Figure 4.8 Trans and cis peptide bonds. The trans form is strongly favored because of steric clashes in the cis form.
Now with any double-bond there are two different orientations that that double- bond can take, cis and trans. And, you can see on the left the trans conformation of this peptide bond keeps the amino acid side groups farther away from each other. Remember these amino acid side groups are marked by green spheres but they could be quite large. And if that peptide bond were to rotate 180 degrees it would bring those amino acid side chains very close together and that is a difficult place for them to be because there is just not enough space for all of the side chains - so you will typically find the peptide bonds in the trans configuration. But they are somewhat flexible and in certain circumstances you might find them in a different configuration.
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Model of Trans and Cis Peptide Bonds
• Most peptide bonds are in the trans configuration so as to minimize steric clashes between neighboring R groups.
Figure 4.9 Rotation about bonds in a polypeptide. The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds. (A) Phi (ø) is the angle of rotation about the bond between the nitrogen and the a-carbon atoms, whereas psi (ψ) is the angle of rotation about the bond between the carbonyl carbon and the a-carbon atoms. (B) A view down the bond between the nitrogen and the a- carbon atoms. The angle ψ is measured as the rotation of the carbonyl carbon attached to the a-carbon atom: positive if to the right, negative if to the left. (C) The angle ψ is measured by the rotation of the amino group as viewed down the bond from the carbonyl carbon to the a-carbon atom: positive if to the right, negative if to the left. Note that the view shown is the reverse of how the rotation is measured, and consequently, the angle has a negative value.
So you can’t usually get much rotation around the peptide bond, but you get free rotation around the other two bonds and those are labeled the phi bond and the psi bond. Those are both attached to the alpha carbon and those can actually rotate more or less freely. However, you know, there are some restrictions based on things banging into each other, etc.
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Model of Rotation About Bonds in a Polypeptide
• Rotation is permitted about the N–Cα bond [the phi (Φ)bond) and about the Cα- carbonyl bond (the psi (ψ) bond].
• The rotation about the Φ and ψ bonds, called the torsion angle, determines the path of the polypeptide chain. Not all torsion angles are permitted.
You can see on this diagram what these numbers mean - if you are looking down the nitrogen-alpha carbon bond, as in the picture on the left (that’s B), you can see there is an angle between the carbonyl on the close side and the carbonyl on the far side, and that angle is called the phi angle. In the other diagram you are looking down from the alpha carbon to the carbonyl group and you can see that there is an angle between the two nitrogen bonds, and that is the PSI angle {oops! I said “phi”}.
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Figure 4.10 A Ramachandran diagram showing the values of Φ and ψ. Not all Φ and ψ values are possible without collisions between atoms. The most favorable regions are shown in dark yellow on the graph; borderline regions are shown in light yellow. The structure on the right is disfavored because of steric clashes.
So there are only certain combinations of phi and psi angles that will work such that the atoms are not in conflict with each other, and those are illustrated here in this diagram which we call a Ramachandran plot which has the phi and psi angles from - 180 to +180, so that is a full 360 degrees. As you can see if the phi angle is around - 120 lets say, then the psi angle can be -60. That brings us into that dark green area. Similarly, we can flip that psi angle around 180 degrees and it would be at +120 which would be in the dark green area as well. That area is much more favored than other things. But if you find a combination of phi and psi that are in the white area, that is not likely to occur.
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A Ramachandran Diagram Showing the Values of Φ and ψ.
Nice video: https://www.youtube.com/watch?v=Kewhg5spUjs
Not all Φ and ψ values are possible without collisions between atoms. The most favorable regions are shown in dark yellow on the graph; borderline regions are shown in light yellow. The structure on the right is disfavored because of steric clashes.
Secondary structure is the next level of organization found in proteins, and it is generally created by hydrogen bonding between nearby amino acid residues. There are two main examples of secondary structure, and those are the alpha helix and the beta sheet.
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Section 4.2 Secondary Structure: Polypeptide Chains Can Fold into Regular Structures
• Secondary structure is the three-dimensional structure formed by hydrogen bonds between peptide NH and CO groups of amino acids that are near one another in the primary structure.
• The α helix, β sheets, and turns are prominent examples of secondary structure.
Figure 4.11 The structure of the alpha helix. (A) A ribbon depiction shows the α- carbon atoms and side chains (green). (B) A side view of a ball-and-stick version depicts the hydrogen bonds (dashed lines) between NH and CO groups. (C) An end view shows the coiled backbone as the inside of the helix and the side chains (green) projecting outward. (D) A space-filling view of part C shows the tightly packed interior core of the helix.
“The alpha helix is a tightly coiled rod-like structure with the R groups bristling out from the axis of the helix.” That is a pretty good description, and you can see in the pictures on the left that the amino acid side chains are sticking out from the center spiral, which is represented there as a ribbon. A full atomic model would be in the center and there you can see the hydrogen bonding between the carbonyl groups and the amino hydrogens. It’s these hydrogen bonds that stabilize the structure. On the top right you can see a look down the axis, and on the bottom right is a space filling representation of that same axis.
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Structures of the Alpha Helix The α-helix is a tightly coiled rod-like structure, with the R groups bristling out from the axis of the helix. All of the backbone CO and NH groups form hydrogen bonds except those at the end of the helix.
Now in those previous images the side groups were all represented by green balls, but I thought I would show you a picture of what it might look like in an actual molecule because the side groups are all present. I see there a methionine, with the yellow, some glutamine, you know, various amino acids sticking out of the side, and so the alpha helix does not have a completely cylindrical shape but it is generally long and thin and that is what you can see from these images.
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Figure 4.12 The hydrogen-bonding scheme for an α helix. In the α helix, the CO group of residue i forms a hydrogen bond with the NH group of residue i + 4.
Let’s talk about that hydrogen bonding a little bit. It turns out that in an alpha helix (and that is the most common form of helix that is formed – there are a few other ones but we might not even touch on that very much, but in an alpha helix...) we see that each oxygen is bound to a hydrogen on the residue that is four residues down from it. You might be able to see this if you look carefully at the picture above which was stolen from a few slides ago, but it is difficult to see.
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The Hydrogen-Bonding Scheme for an Alpha Helix
In the α helix, the CO group of residue i forms a hydrogen bond with the NH group of residue i + 4.
Figure 4.13 Schematic views of alpha helices. (A) A ribbon depiction. (B) A cylindrical depiction.
So in schematic pictures of proteins, alpha helices will often be represented in this ribbon form on the left, or in just a solid rod/cylinder on the right.
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Schematic Views of Alpha Helices
Figure 4.14 A largely α-helical protein. Ferritin, an iron-storage protein, is built from a bundle of α helices. [Drawn from 1AEW.pdb.]
This is a depiction of ferritin, an iron storage protein, which is largely a bundle of alpha helices. This is actually horse ferritin and the code of this structure is 1AEW. That is the protein data bank code. So, the protein data bank is actually an extremely useful resource and I encourage you to check it out. There is a lot of good information there and tutorials and structures of all of the known proteins. I have provided you a link at the bottom of the page which I recommend you take a look at. If you want to get more deep into protein structure you might also consider using a program called UCSF Chimera as a molecular viewer. It is a free program that you can download and it is very powerful as far as letting you visualize proteins in different ways.
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Model of Ferritin, a Largely Alpha-Helical Protein
Download structure from https://www.rcsb.org/structure/1AEW. Also, consider UCSF Chimera as a molecular viewer
A largely α-helical protein. Ferritin, an iron-storage protein, is built from a bundle of α helices. [Drawn from 1AEW.pdb.]
Figure 4.15 The structure of a b strand. The side chains (green) are alternatively above and below the plane of the strand. The bar shows the distance between two residues.
Beta sheets are another common secondary structure and instead of being wrapped up in a helix, the beta sheets are extended.
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Beta Sheets Are Stabilized by Hydrogen Bonding Between Polypeptide Strands
The structure of a b strand. The side chains (green) are alternatively above and below the plane of the strand. The bar shows the distance between two residues.
• The β sheet is another common form of secondary structure.
• Beta sheets are formed by adjacent β strands.
• In contrast to an α helix, the polypeptide in a β strand is fully extended.
Figure 4.16 Antiparallel and parallel β sheets. (A) Adjacent β strands run in opposite directions. Hydrogen bonds (green dashes) between NH and CO groups connect each amino acid to a single amino acid on an adjacent strand, stabilizing the structure. (B) Adjacent β strands run in the same direction. Hydrogen bonds connect each amino acid on one strand with two different amino acids on the adjacent strand.
Beta sheets are connected to each other by hydrogen bonds, and you can see the hydrogen bonds between the oxygens and the hydrogens represented here as green dashed lines. The boxed areas represent individual amino acids. So there are a few different ways that these polypeptide strands can come together. One is antiparallel which you see at the top, which means that they are parallel to each other but going in opposite directions. In that configuration, you can see that amino acids bond directly to other amino acids that are straight across from them. On the bottom you see the parallel orientation where they are both oriented in the same way, and the amino acids interact with the next amino acid down the line, kind of forming a daisy chain.
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Structure of Antiparallel and Parallel Beta Sheets
• Hydrogen bonds link the strands in a β sheet.
• The strands of a β sheet may be parallel, antiparallel, or mixed.
Figure 4.17 The structure of a mixed β sheet.
You can also have beta sheets that are combinations of parallel and antiparallel chains.
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Structure of a Mixed Beta Sheet
Figure 4.18 A twisted β sheet. (A) A schematic model. (B) The schematic view rotated by 90 degrees to illustrate the twist more clearly.
Beta sheets can be flat or twisted. They can be quite twisted, but you would rarely see one that is actually flat - I mean, there is usually some sort of twist to them. This is one picture that shows from the side view one beta sheet and then the end view on the right (B) how it twists, sort of counterclockwise. I guess that would be a left hand twist to the beta sheet.
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Model of a Twisted Beta Sheet
Figure 4.19 A protein rich in β sheets. The structure of a fatty acid-binding protein. [Drawn from 1FTP.pdb.]
Here we see a common motif of a beta sheet twisted into a barrel form or a cylinder. You can see also an alpha helix, a couple of them, at the top.
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Model of a Protein Rich in Beta Sheets
Figure 4.20 The structure of a reverse turn. (A) The CO group of residue i of the polypeptide chain is hydrogen bonded to the NH group of residue i + 3 to stabilize the turn. (B) A part of an antibody molecule has surface loops (shown in red). [Drawn from 7FTP.pdb.]
Hydrogen bonding can also create secondary structures in other ways such as when there is a short loop in the amino acid chain. Two amino acids, as we see here, i and i+3, are combined - that stabilizes that turn. You can see on the right what those loops at the top would look like (those are in red) and there can be hydrogen bonds between the loops as well.
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Polypeptide Chains Can Change Direction by Making Reverse Turns and Loops
Figure 4.21 An α-helical coiled coil. (A) Space-filling model. (B) Ribbon diagram. The two helices wind around each other to form a superhelix. Such structures are found in many proteins, including keratin in hair, quills, claws, and horns. [Drawn from 1CIG.pdb.]
Now, I debated whether or not to include this, but it’s actually kind of cool, so I decided to include it here. Coiled coils are actually a phenomenon that you see. They are not a secondary structure per say, but they are composed of alpha helices that are winding around each other.
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Fibrous Proteins Provide Structural Support for Cells and Tissues
• α-Keratin, a structural protein found in wool and hair, is composed of two right-handed α helices intertwined to form a left-handed superhelix called a coiled coil. The helices interact with ionic interactions or van der Waals forces.
• α-Keratin is a member of a superfamily of structural proteins called coiled-coil proteins. Other members of the family include some cytoskeleton proteins and muscle proteins.
Collagen is a structural protein that has three intertwined helical polypeptide chains. It has a very interesting structure - because these chains have to be very close together, every third residue is a glycine (remember glycine does not take up much space, it’s just a hydrogen). Then it is often followed by two prolines, which bend the molecule, and therefore, make these able to intertwine.
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Collagen
• Collagen is a structural protein that is a component of skin, bone, tendons, cartilage, and teeth.
• Collagen consists of three intertwined helical polypeptide chains that form a superhelical cable. The helical polypeptide chains of collagen are not α helices.
• Glycine appears at every third residue, and the sequence Gly-Pro-Pro is common.
Figure 4.22 The amino acid sequence of a part of a collagen chain. Every third residue is glycine. Proline and hydroxyproline also are abundant.
So, here you see the sequence of the collagen chain. Every third residue is a glycine - those are in red. That is invariant. In between the glycines you see a lot of prolines, the Pro and Hyp, in green. Hyp is hydroxyproline, which is a variation on proline but still has that ring-like structure that gives a bend in the polypeptide chain.
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The Amino Acid Sequence of a Part of a Collagen Chain
Figure 4.23 The conformation of a single strand of a collagen triple helix.
Here’s a schematic that shows you what that polypeptide chain would look like.
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Model of the Conformation of a Single Strand of a Collagen Triple Helix
Figure 4.24 The structure of the protein collagen. (A) Space-filling model of collagen. Each strand is shown in a different color. (B) Cross section of a model of collagen. Each strand is hydrogen-bonded to the other two strands. The α-carbon atom of a glycine residue is identified by the letter G. Every third residue must be glycine because there is no space in the center of the helix. Notice that the pyrrolidine rings are on the outside.
So you can see here in B that the three chains are held together by hydrogen bonds. Those are the little dotted lines between them. And there is not space in the center for any larger amino acids, so that’s why they all have to be glycine. This helix then would have three amino acids per turn {I’m actually not sure about this statement, even though I looked it up. So don’t worry about it.}
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Collagen consists of three intertwined chains
• The helices in collagen are not stabilized by hydrogen bonds. Rather, they are stabilized by steric repulsion of the pyrrolidine rings of proline. The three intertwined chains interact with one another with hydrogen bonds.
• The interior of the superhelical cable is crowded, and only glycine can fit in the interior.
So problems with your collagen structure can make you sick. One of these conditions, Osteogenesis Imperfecta, or brittle bone disease, occurs when one of those glycines gets replaced by another amino acid. And then the collagen can not wind up as effectively, and the collagen ends up being weaker and your bones end up being more brittle. Now there’s a ton of places, a lot of mutations, that can replace one of these glycines. There’s probably hundreds of glycines in each molecule so each one of these mutations might represent a different variation of the gene and thus a variation of the disease. So these things might be slightly different but they all have similar symptoms.
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Clinical Insight
CLINICAL INSIGHT Defects in Collagen Structure Result in Pathological Conditions
• Osteogenesis imperfecta, or brittle bone disease, occurs if a mutation results in the substitution of another amino acid in place of glycine.
Now remember hydroxyproline, we talked about? That’s a very important part of the collagen, and if you don’t have the hydroxyproline, well, you start getting sick. As it turns out, we make hydroxyproline out of proline in our bodies. Part of that process is mediated by vitamin C. And if you don’t get enough vitamin C, you get scurvy, which means you can’t make enough hydroxyproline and therefore your collagen becomes destabilized and your body literally starts to fall apart until you bleed to death.
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Clinical Insight
CLINICAL INSIGHT Defects in Collagen Structure Result in Pathological Conditions
• Hydroxyproline, a modified version of proline in which a hydroxyl group replaces a hydrogen, is important for the stabilization of collagen.
• Vitamin C is required for the formation of hydroxyproline. A lack of vitamin C results in scurvy.
Now onto the third level of organization in proteins, that is tertiary structure. And, all of these beta sheets and alpha helices come together to form globular proteins, which we’ve seen some examples of already.
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Section 4.3 Tertiary Structure: Water-Soluble Proteins Fold into Compact Structures
• Tertiary structure refers to the spatial arrangement of amino acids that are far apart in the primary structure and to the pattern of disulfide bond formation.
• This level of structure is the result of interactions between the R groups of the peptide chain.
Figure 4.4 Cross-links. The formation of a disulfide bond between two cysteine residues is an oxidation reaction.
One thing that links proteins together, forming long-distance interactions between different parts of the protein, are cysteine disulfide bonds. And you can see here on the left when two cysteines come together they create a bond between the sulfurs, and that is a strong bond that will help to organize the protein.
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• In some proteins, the polypeptide chain can be cross-linked by disulfide bonds.
• Disulfide bonds form by the oxidation of two cysteines.
Figure 4.5 Amino acid sequence of bovine insulin.
An excellent example of that would be insulin, which we have here. This is bovine insulin, but human insulin is very similar. And you see how there are actually two separate chains in insulin that are connected with these disulfide bonds. Actually, insulin starts out as one chain and then the disulfide bonds are formed, and then proteases come and break it apart into two chains. But the disulfide bonds keep it together and end up being the important hormone that we know {insulin}.
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Amino acid sequence of bovine insulin.
Proteins Have Unique Amino Acid Sequences Specified by Genes
Figure 4.31 Amino acid sequence of bovine ribonuclease. The four disulfide bonds are shown in color. [After C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 235(1960):633–647.]
Another example here would be the sequence of bovine ribonuclease - just some random protein. But as you can see bovine ribonuclease contains four disulfide bridges between various parts of the chain that hold it in its particular order. This is an example of using the one-letter representations of the amino acids, in case you were wondering what these letters mean.
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Figure 4.25 The three-dimensional structure of myoglobin. (A) A ribbon diagram shows that the protein consists largely of α helices. (B) A space-filling model in the same orientation shows how tightly packed the folded protein is. Notice that the heme group is nestled into a crevice in the compact protein with only an edge exposed. One helix is blue to allow comparison of the two structural depictions. [Drawn from 1A6N.pdb.]
So, we are going to use myoglobin as an illustration of tertiary structure, and as you can see here on the left, there are several alpha helices in myoglobin. This is the oxygen-carrying molecule in your muscles. All of these secondary structures, these alpha helices, bundle together to form a globular protein, which you can see clearly on the right. The blue alpha helix on the left, is represented also in blue on the right. That’s not to say that it’s anything specifically different than the rest of the protein, it’s just so you can trace the image of the alpha helix on the left and see how it turns out on the right with all of the amino acids added to it. Another important part of this protein is the heme group, which is a prosthetic group and we will be talking about those in detail later, but this heme group is nestled right in the little cleft there inside the myoglobin protein. The cleft is very perfectly suited for that heme group, and of course in that heme group is an iron atom which helps carry around the oxygen.
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Myoglobin Illustrates the Principles of Tertiary Structure
• Globular proteins such as myoglobin form complicated three-dimensional structures.
• Globular proteins are very compact. There is little or no empty space in the interior of globular proteins.
Figure 4.26 The distribution of amino acids in myoglobin. (A) A space-filling model of myoglobin, with hydrophobic amino acids shown in yellow, charged amino acids shown in blue, and others shown in white. Notice that the surface of the molecule has many charged amino acids, as well as some hydrophobic amino acids. (B) In this cross-sectional view, notice that mostly hydrophobic amino acids are found on the inside of the structure, whereas the charged amino acids are found on the protein surface. [Drawn from 1MBD.pdb.]
Here’s that same protein, myoglobin, but in these images the hydrophobic amino acids are shown in yellow and the hydrophilic amino acids are shown in blue. That’s kind of an important point to make right now, is that the colors of these amino acids and proteins are not necessarily standardized, so it’s important to read the legends of figures and find out exactly what the color is, what the colors mean. In the left hand side, you see an overall picture of the myoglobin and you can see along the outside you see a lot of these blue residues, these are the hydrophilic residues, and naturally they are on the outside to interact with the water, where they like to be. On the right is a cross section of myoglobin, so you can see what’s going on in the middle of the myoglobin. And as you can see, it’s mostly these hydrophobic residues. The hydrophobic parts are actually very important for the folding of the protein. They tend to accumulate in the center of the proteins, away from the water, and that tendency is one of the most important factors in protein folding.
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• The interior of globular proteins consists mainly of hydrophobic amino acids.
• The exterior of globular proteins consists of charged and polar amino acids.
Here is a slightly different depiction of, I believe this is hemoglobin actually, but if you check the link at the bottom (http://www.rcsb.org/pdb/101/motm.do?momID=41) you will see an excellent animation on that page of the binding of oxygen to the iron in the hemoglobin and the changes that occur in the protein when that happens. It is important to know that these protein shapes are not static, they do move around and they can be highly dynamic. I will get to that in a minute.
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Figure 4.28 Protein domains. The cell-surface protein CD4 consists of four similar domains. [Drawn from 1WIO.pdb.]
Now another feature of tertiary structure is that a single polypeptide chain can actually bundle into separate units and these protein units are called domains. Often the domains have each a specific function and they work together to perform an overall action of the protein. But here we can see the red, yellow, blue, and orange domains that are formed by this single polypeptide chain. These are all beta sheet mediated, they look like beta sheets mostly in the secondary structure, but they are clearly separated into four separate domains.
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The Tertiary Structure of Many Proteins Can Be Divided into Structural and Functional Units
• Some proteins have two or more similar or identical compact structures called domains.
Figure 4.29 Quaternary structure. The Cro protein of bacteriophage λ is a dimer of identical subunits.
And finally, we have quaternary structure, which is the assembly of different proteins into a single functional unit. That is something that happens all the time in proteins. These proteins work together to form these complex engines, and the way that they fit together is called their quaternary structure. So on the bottom here we have a dimer, actually two separate identical proteins, that interact with each other and form a functional unit called a dimer. Like I said, this is extremely common. In fact you are more likely to find proteins in these structures than you are to find them alone in the cytoplasm.
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Section 4.4 Quaternary Structure: Multiple Polypeptide Chains Can Assemble into a Single Protein
• Many proteins are composed of multiple polypeptide chains called subunits. Such proteins are said to display quaternary structure.
• Quaternary structure can be as simple as two identical polypeptide chains or as complex as dozens of different polypeptide chains.
Cro protein
Figure 4.30 The a2β2 tetramer of human hemoglobin. The structure of the two identical a subunits (red) and the two identical β subunits (yellow). (A) The ribbon diagram shows that they are composed mainly of a helices. (B) The space-filling model illustrates the close packing of the atoms and shows that the heme groups (gray) occupy crevices in the protein. [Drawn from 1A3N.pdb.]
Here is another example of quaternary structure. Hemoglobin is actually four units that are … not quite identical, there are two hemoglobin alpha or hemoglobin A, and two hemoglobin B, and you can see those are represented as the red units and the yellow units. But they come together to form a four-membered hemoglobin, and these proteins work together to perform the functions of hemoglobin cooperatively.
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Model of a2β2 Tetramer of Human Hemoglobin
ALPHA
BETA
And how do these proteins fold to reach these structures? Well, they fold progressively. They will start with local folding and then, after the local parts have folded up the secondary structures will interact with each other and start to form tertiary and quaternary structures.
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Section 4.5 The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure
• Protein folding also occurs by cumulative selection. Partly correct folding intermediates are retained because they are slightly more stable than unfolded regions.
Proteins Fold by the Progressive Stabilization of Intermediates Rather Than by Random Search
Figure 4.35 Folding funnel. The folding funnel depicts the thermodynamics of protein folding. The top of the funnel represents all possible denatured conformations—that is, maximal conformational entropy. Depressions on the sides of the funnel represent semistable intermediates that may facilitate or hinder the formation of the native structure, depending on their depth. Secondary structures, such as helices, form and collapse onto one another to initiate folding. AFTER D. L. NELSON AND M. M. COX, LEHNINGER PRINCIPLES OF BIOCHEMISTRY, 5TH ED. (W. H. FREEMAN AND COMPANY, 2008), P. 143.]
So, this is a diagram that is in your textbook, called the folding funnel, and it’s a pretty good concept. At the top we have high energy, so that would be if you pulled the protein apart into its linear form, so, just it’s a string of amino acids. It would require some energy to do that - you would have to put energy into it to do that. That would be its state of highest entropy, where all of the phi / psi angles are free to wobble and turn in any direction they want. But if you let go of the ends of that string, then it will start to collapse, and the protein amino acids will start interacting with each other, hydrogen bonding will occur, creating alpha helices and beta sheets, and right around there you might find yourself in this molten globule state that you see in the middle. The entropy has decreased and the energy in the protein has also decreased, and you’ve got sort of an intermediate state. As time passes, this intermediate state will get more and more compact, as the amino acids find each other in more and more
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Diagram for Folding Funnel Model
tight bonding configurations, until finally you reach the bottom of the well, what we call the native structure, which is the structure of the lowest energy.
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This is a very similar drawing that shows you examples on the right hand side, between the unfolded state, the molten globule state, and the native state.
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But I think this is probably the best illustration of the principle. You start up at the top with unfolded proteins, and there are several different conformations that these unfolded proteins can fall into. Now you’ll notice in this diagram that there are several wells, and small wells as well as large wells. Each one of those represents a local energy minimum - so that’s a state the protein can fall into as its energy gets dispersed into the surrounding medium, and it will stay there for a little while because locally that is a favorable energy state. But as time goes by, energy comes and goes, and will eventually push it back up out of that well and then it will be able to fall into other wells, until it gets to a point where it’s at a very low point, such as you see in the native state, or there in the amorphous aggregate state or the amyloid fibrils state. Those wells are so low that the protein tends to just stay in those states. Not to say that it’s a permanent position, because the native state is stable but there is still a lot of energy fluctuation going on, so it can actually be pushed out of the native state - and sometimes that does happen, especially in diseases such as Huntington’s Disease or Alzheimer's Disease, where the native state falls apart and the protein falls into one of these amorphous states - or even worse, one of these amyloid fibril states. An amyloid fibril state is extremely stable and once you’ve found yourself in that condition, you’re unlikely ever to come out of it. Something that prevents that from happening is the action of chaperones. They interact with partially folded proteins and prevent them from going into amorphous aggregates, but encourage them to go back toward the native state. Some sort of
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mutation in your chaperones, though, or some other condition that makes your chaperones non-functional, can lead to severe disease conditions.
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Interestingly, a lot of these conditions are neurological conditions - I think because the brain is one of the most sensitive parts of the body. You just start to see the effects in the brain earlier than you see them in muscles or skin tissue. Alzheimer's disease, Parkinson’s disease, Huntington’s disease, these are all failures of protein folding. Huntington’s disease is in fact the subject of my graduate thesis and I have a great deal of information on that and these amyloidoses. If you’d like to learn more about them, please feel free to ask me any questions.
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Clinical Insight: Protein Misfolding and Aggregation Are Associated with Some
Neurological Diseases
CLINICAL INSIGHT Protein Misfolding and Aggregation Are Associated with Some Neurological Diseases
• Amyloidoses are diseases that result from the formation of protein aggregates, called amyloid fibrils or plaques.
• Alzheimer disease is an example of an amyloidosis.
• Huntington Disease is another, which I did my graduate research on.
Figure 4.38 The protein-only model for prion-disease transmission. A nucleus consisting of proteins in an abnormal conformation grows by the addition of proteins from the normal pool.
Prion diseases are another protein folding disease. Remember how I was telling you about the chaperones, which encourage the protein to fold in the correct way - well, as it turns out, with a prion and also with several of these other protein folding diseases, once a protein is folded in the incorrect way, it sort of acts as a chaperone and encourages the other proteins (that are folded correctly) to fold in the incorrect way, and so leads to a sort of chain reaction and all of the good proteins start to disappear, and these large clumps of protein start to appear that interfere with cellular processes. It’s because of these chaperone-like properties of these misfolded proteins that prion diseases are contagious. If there is a misfolded protein in a cow, and you eat it, then it gets into your body and encourages your body’s native proteins to misfold. These aggregates of misfolded protein are extremely difficult to get apart. In fact, in the laboratory we have a difficult time prying them apart because they are very stable.
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The Protein-only Model for Prion-disease Transmission
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The Alpha Helix Is a Coiled Structure Stabilized by Intrachain Hydrogen Bonds
• The α helix is a tightly coiled rodlike structure, with the R groups bristling out from the axis of the helix.
• The CO group of each amino acid forms a hydrogen bond with the NH group of the amino acid that is situated four residues ahead in the sequence. All of the backbone CO and NH groups form hydrogen bonds except those at the end of the helix.
• Essentially all α helices found in proteins are right-handed.
DID YOU KNOW? Screw sense refers to the direction in which a helical structure rotates with respect to its axis. If viewed down the axis of a helix (N terminus to C terminus), the chain turns in a clockwise direction; it has a right-handed screw sense. If turning is counterclockwise, the screw sense is left- handed.