Arking:JCAOligoTutoria25

Tour of Structural Biology

This tutorial is designed to introduce you to protein structure. We’ll go through the tradition stuff which hopefully you’ve already seen, then we’ll look at some diverse structures of interest, and then we’ll get into active sites and see how they work. At the end, we’ll have a quiz.

Two books you might want to check out that go into greater detail into the basics of structural biology including a tour of many different protein structures are:

Download DeepView

This tutorial will use a freeware program called DeepView. If you skipped the "DeepView Basics" tutorial, you can download it here: http://spdbv.vital-it.ch/disclaim.html

Download Files

This tutorial refers to several PDB files which can be downloaded here.

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The Basics: alpha helices, beta sheets, and hydrophobic cores

There are 4 levels of protein structure: primary (the polypeptide sequence), secondary (alpha helices and beta strands), tertiary (folds), and quaternary (protein-protein interactions). To see how these work, let’s open up “alpha_helix.pdb” in DeepView. It’s a crystal structure of an alpha helix. It will throw up some messages when you open files—just close them.

Recap of DeepView controls

You should see a structure on the screen. If you do not see the control panel, go under Wind > Control Panel to open it up. The control panel shows all the residues in the structure. The first column (here all A’s) is the chain. If there were two polypeptides, or say a protein and a DNA, you’d see more letters. The next column shows whether there is secondary structure present. You’ll see here a bunch of h’s—those mean helices, or alpha helices. The next column are the residues and their position numbered from the N-terminus. You’ll notice that this starts with 3—this is fairly common with protein structure. Often the N and C-termini of a protein are unstructured and do not give clear electron density. In this instance, I chopped an alpha helices out of a larger protein and that is why you only see 15 amino acids. The next column is yes/no as to whether DeepView shows the atom coordinates for the residue. Try clicking on some of them. You’ll notice that clicking one of the v’s will make various sticks disappear. You can click on the same spot again to make them come back. The next column determines whether the side chain is displayed or not. Try clicking off one of those. You’ll notice that the side chains disappear, but the backbone stays lit up. The next column are labels. Try clicking in the white area next to GLN8. You’ll see “Gln8” show up on the screen near the residue. The next column is the space-filling view of the residue The little triangle above it can be clicked on and it allows you to toggle between different types of space-filling views. The next column is the ribbon view. This gives the traditional Richardson view of the structure. Finally, you have the color column and a triangle to the right of it. You can toggle between coloring the side chain, the ribbon, the backbone, and so on by choosing the one you want from the triangle and then clicking on the square under “col” and choosing a color. If you just hit “cancel” from the color window that pops up, it will use CPK coloring which means red for oxygen, blue for nitrogen, yellow for sulfur, and white for carbon. You can play with the control panel to familiarize yourself with turning things on and off.

Now let’s play with the navigation controls. At the top of the screen you’ll see some icons:

The first one centers the currently-selected residues in 3 dimensions and scales it to fit your window. The next one is the hand symbol. First click on it to highlight it, then click on the structure and you can drag the view left and right. The next one allows you to scale the view by clicking and moving the mouse up and down. You can achieve the same result by holding down the left and right clicks and dragging the mouse up and down. The next one rotates the view with left clicks when highlighted. It rotates the view around the current center point. You can re-center the entire thing by clicking on the first re-center and scale button. So, practice moving the molecule around on your screen and get comfortable looking at it.

You can also put the view in stereo mode by going under Display > Stereo View. This gives me a headache, so I never use it, but if you want to ruin your eyes, turn this on and click on the rotate button. Look at the screen cross-eyed, and a third version of the structure will appear in the middle (an illusion, of course). Staring at the middle structure, move the mouse around, and it should pop out at you as 3D. More sophisticated structure viewing tools give you special goggles to look at things in 3D, but if you just constantly wiggle the rotation around you’ll get used to looking at things in 2D. Alright, those are the basic viewing tools in DeepView. We’ll play with more of them later, but let’s learn about structure.

Examination of alpha helix secondary structure

Go under Wind > Ramachandran Plot to pull up the window. If you aren’t familiar with this plot, what it tells you is the phi and psi angles of all the backbone bonds. The “Calpha” positions of all the amino acids in a structure in combination with the psi and phi angles define the backbone positions of the structure. You’ll notice there are several distinct zones colored in the plot. What these refer to are the “allowed” angles for polypeptides. Basically, outside of these zones you are getting an angle of the bonds that doesn’t work electronically or sterically. Go into the control panel. You’ll notice some things are black and some things are red. Click on “GLN8”—it will turn red and everything else turns black. Now click ctrl-A to select everything—they all turn red again. Now do that same thing while looking at the Ramachandran plot. You’ll see little white dots appearing and disappearing as you click. Each dot represents a different residue in the protein, and it is plotting it’s psi and phi angles. You’ll notice that all these residues have angles very similar to one another within a little yellow zone. This is because are current structure is all alpha helix. Secondary structure is defined by stretches of residues with similar psi and phi angles. So, it’s no coincidence that all these residues have similar angles. You can close the Ramachandran plot now. Let’s look a little closer at the structure. Click around on the control panel to make it look like the image below. If you hold shift while clicking, you’ll perform the change you want to the entire column rather than just a single cell—that speeds things up. You can also click and drag to change multiple cells at once.

Click on Tools > Compute H-bonds. Little green lines should pop up. These represent calculated hydrogen bonds. These are calculated by the program whenever appropriate hydrogen bond donor and acceptors are within a certain distance from each other and an acceptable orientation for H-bonding. Rotate the view around and center it to orient yourself, and then shift-click on the ribn column to get rid of it. Since we have the side chains off, you are just looking at the backbone, but notice how the oxygens of all the backbone carbonyl groups hydrogen bond to alpha amino groups on the N+4 residues. Now orient your view so that you are looking down the barrel of the alpha helix. Click on the word “side” in control panel. This will light up the side chains for the selected residues (8 through 15). You’ll notice that they all point out at various angles towards the outside of the helix. So, that’s the alpha helix—you have a spiral made up of the backbone with side chains sticking straight out from it. Now, let’s look at the whole protein.

Examining the tertiary structure of an all-alpha helix protein

Open up 2JHO.pdb and turn off the “show” and “side” for everything, and turn on “ribn” for everything. So, this is a myoglobin from sperm whale. It’s an all alpha-helix protein. Open up the Ramachandran Plot. Select all the residues in the control panel and notice where they show up—a lot of alpha helix, huh. Now select resdues 10-34 and turn on show, side, and rib. Center your view. Look at your Ramachandran Plot—you’ll notice that only one residue isn’t in the alpha-helix angle zone. It’s asp20. Now, turn off all side-chains except asp20. Turn on the label if you can’t find it, but you’ll see its right at the junction between the two alpha helices. So, in protein domains that are “all alpha helix”, you’ll get loops that have phi and psi angles outside of the alpha helix region.

Let’s look at the whole thing again. Select everything, turn on ribn only. What you see is about 7 alpha helices piled on top of each other. Let’s look at folding now. Select all and turn on “show”, “side”, and “::v”. Click on the color triangle and select “backbone and side”. Click on the word “col” and select one of the orange colors and say ok. You should see a big mess of orange dots now (and no other colors). Alright, now select all the hydrophobic residues by doing Select > Group Property > Non Polar. With those selected, click on “col” and choose one of the greens. Now, the screen looks really complicated.

Examining the hydrophobic core

Let’s put it in “slab mode” to simplify it: Display > Slab. You’re now looking at a cross-section of the structure. You can use your rotate tool to spin things around in 3D. Take a look at it from multiple views. Notice a pattern? In general, you’ll see that almost exclusively green dots are in the middle, and orange dots on the outside. That’s basically what is driving proteins to fold into tertiary structures—you have a hydrophobic core being pushed to the middle, and mostly polar residues being brought to the surface. You’ll notice, though, that there are still a variety of hydrophobic patches on the surface. That’s pretty typical. The important thing for folding is to have a tightly packed, all hydrophobic core. I recommend you go through this procedure of lighting up the hydrophobic core for all the proteins we look at in this tutorial to see the degree to which this is true. Maintaining the current slab view, also turn on ribn and rotate the structure around. You’ll see now how alpha helices interact with each other. Each alpha helix extends side chains from it, and the hydrophobic ones orient towards the core while the polar ones orient themselves outward.

Examining the tertiary structure of an all-beta sheet protein Let’s now turn to a beta strand. Open up “2IZJ.pdb”. Turn on “show” and “ribn” for 26-35 and center your view. Open up the Ramachandran Plot. You’ll notice that all the phi and psi angles are again in a similar region, but it is a different region than the alpha helices. The backbone of a beta strand is all stretched out making for a long flat peptide. Go Tools > Compute H-Bonds. You’ll notice that nothing popped up. There are no hydrogen bonds within a beta strand! Let’s expand the view: select 17-45. Turn on “show” and “ribn”. Now we have 3 beta strands in view and you should see some green hydrogen bonds. So, in beta sheets you get hydrogen bonding between the backbone carbonyls and amino groups from adjacent strands. Now, let’s also turn on the sidechains “side”. Select all. Using the color triangle, make “backbone + side” orange. Wiggle around your rotation, and you’ll see that the side chains all point above and below the beta sheet.

Now, put everything into view, turn on show, side, ::v, and ribn on. Color all the backbones and side chains orange, select the hydrophobic residues, and color them green. Now wiggle around your screen in slab view. The hydrophobic core of this protein (streptavidin) is a little less apparent than the myoglobin structure, but check out this view looking down the barrel:

So, you get one face of the beta sheets jutting out hydrophobic residues towards the core, and the other face jutting out polar residues towards the solvent. You’ll notice there are some polar residues inside the core of this protein. That seems a little strange. The reason for it is that this protein binds a small molecule (biotin) by stuffing the small molecule inside the barrel. The non-hydrophobic amino acids will interact with the non-hydrophobic moieties present in the ligand.

Inverting the hydrophobic core

Let's take a look at the integral membrane protein bacteriorhodopsin and see how it folds. In the membrane, you have an aqueous environment "above" and "below" the protein, but hydrophobic residues all around it. So, how does this play out in terms of folding? Open up 2I20.pdb. I've already colored all the hydrophobic residues green, the lipid residues from the membrane around it dark green, and everything else is yellow:

Notice how the outer rim of all the alpha helices is hydrophobic and the internal core is mostly hydrophilic. Spin the files around on the screen and look at the top and bottom of the barrel. Notice that the residues capping the barrel are more hydrophilic. Make sense? You are still getting folding by driving hydrophobic residues to a hydrophobic environment, and hydrophilic residues towards an aqueous environment.

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Motifs and Domains

A domain is a discrete structural unit that is assumed to fold independently of the rest of the protein

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The Active Site

The thing about proteins that leads us to study them isn't really folding. It's what they do after they fold. Now, of course, the folding is necessary to get to the structure that enables them to do whatever they are going to do, but folding alone doesn't accomplish anything within the cell. What proteins and RNAs do is pretty diverse. On the figure below I've pointed to various interactions that occur within biology. The arrows correspond to physical interactions between biomolecules, and they can be either binding (just sticking together with no change in either's chemical composition) or catalytic (interaction results in a change in modifications to the covalent bonds in one or the other molecule). The one with a dotted line isn't really a means of achieving function in a cell, but all the others are quite common: Proteins can interact with other proteins, RNAs, DNAs, or small molecules. RNA has similar diversity of interactions, but not quite as extensive as protein. Missing from the figure are the carbohydrate polymers and lipid membranes. I guess I'd lump them into the same category as metabolites--from a functional perspective they (as far as we know now!) are passive endpoints in biology. Things certainly interact with and manipulate them, but you don't get carbohydrate-based catalysts.

So, let's take a tour of some of the more famous examples of these interactions. For each one, I've put some questions in red for you to answer as your quiz.

Biotin/Streptavidin

The interaction between biotin (a vitamin) and streptavidin (a bacterially-derived protein) is the strongest protein-small molecule interaction known. There is also a protein called avidin from eggs that binds biotin and is very similar structurally and functionally to streptavidin, yet I digress. The structural basis for this interaction has been investigated, and you can read about it at PMID: 2911722. Let's go through the PDB files and take a look at how it works. There are 2 PDB files you should download: 2IZA and 1STP. The first is the "apo" structure which means that biotin is not bound to the active site.

The "Cost" of Unsatisfied Hydrogen Bonds

A general principle about protein folding deals with the “cost” of unsatisfied hydrogen bonds. Pioneering studies by Richard Wolfenden a long time ago showed that in an aqueous environment, there is a huge energy cost of having unsatisfied hydrogen bonds. So, it is extremely rare to find polar residue sidechains or free water molecules inside a hydrophobic core. This is an important concept in understanding structure. There is very little enthalpic or entropic gain for forming a hydrogen bond—-except in the context of alpha helices and beta sheets where the hydrogen bonding is cooperative. For other hydrogen bonds, the energetic are largely a wash if those hydrogen bonds are satisfied by binding to water or to other amino acid side chains or backbones. There is an enormous energy cost if the hydrogen bonds are not satisfied by any of these. To get another view of the importance of hydrogen bonds, let’s look at a set of 3 crystal structures:

 Nature. 1989 Aug 3;340(6232):404-7.  PMID: 2818726
 Substrate specificity and affinity of a protein modulated by bound water molecules.
 Quiocho FA, Wilson DK, Vyas NK.

Now, the first thing to know is that this protein normally binds arabinose, but it can bind to ther sugars with varied affinities:

 Arabinose 0.98E-7M
 Galactose 2.3E-7M
 Fucose    3.8E-6M

These are “dissociation constants” for the binding. Basically, they are the concentration of small molecule at which 50% of the proteins will be associated with the small molecule. So, smaller numbers mean tighter binding.