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Determining how proteins fold in three-dimensional space was a monumental challenge. Sir William (Lawrence) Bragg and his father, Sir William Henry Bragg, were awarded the Nobel Prize in Physics in 1915 for using X-rays to determine the structure of crystals. British researchers John Kendrew and Max Perutz began using X-ray crystallography in the 1930s to deduce the structure of hemoglobin. They finally succeeded in 1958!
It took 25 years to deduce the structure of hemoglobin from X-ray diffraction patterns, but what took them so long? You've probably seen an image of 'photo 51' – the X-ray diffraction pattern of DNA taken by Rosalind Franklin. The 'dots' on this diffraction pattern are in a nice array, because DNA is a regularly ordered structure (a double helix). Proteins aren't uniform in structure. (Although they consist of uniform secondary structures, these secondary structural elements are arranged differently in three-dimensional space for various proteins.) Therefore they are much more difficult to analyze. And in the 1950s, there were no desktop computers, no laptops, no cell phones – not even calculators. All the mathematical calculations (Fourier transforms) and model building had to be done by hand.
Today, we have high-throughput, automated methods to analyze X-ray diffraction patterns. We also rely on the experience of those scientists who have gone before us and paved the way – both in methodology and in examples of other structures – to guide us into interpreting X-ray diffraction data. So we now know that myoglobin and hemoglobin and leghemoglobin are all similar in structure – and are all part of the 'globin family' of proteins. This tutorial explores the structure and function of some of the members of this protein family.
Myoglobin was the first high resolution protein structure to be determined in 1958 by John Kendrew and Max Perutz. It is a single subunit protein that stores oxygen in the muscles. Myoglobin is what gives meat its red color. Let's explore the structure of myoglobin.
What secondary structures are found in this protein?
The secondary structure consists of alpha helices and beta sheets.
The secondary structure has a parallel beta sheet and no helices.
The secondary structure has an antiparallel beta sheet and no helices.
The secondary structure has alpha helices.
Myoglobin, like all proteins in the globin family, has alpha helices but NO beta strands.
In addition to helices and sheets, proteins often have less structured regions called loops and turns. Turns are typically short regions between two secondary structures. They are stabilized by hydrogen bonds. Loops are typically longer, and, because they are not stabilized by hydrogen bonds, often are involved in movement of a protein (conformational change) when a protein interacts with a substrate or other molecule.
Sperm whale myoglobin; hydrogen bonds are colored green. For clarity, hydrogen bonds in helices are not displayed. PDB ID: 1mbnLook at the backbone regions that are colored white. Predict whether these are loops or turns.
The turns have 1-3 amino acids and the loops have 5 or more amino acids.
All of the white regions are loops because they are stabilized by hydrogen bonds.
All of the white regions are turns because they are stabilized by hydrogen bonds.
The loops have 1-3 amino acids and the turns have 5 or more amino acids.
Myoglobin has several turns that are stabilized by hydrogen bonds. There are no flexible loops involved in conformational changes in this protein.
In addition to protein, myoglobin contains a prosthetic group called heme. Prosthetic groups help to stabilize protein structure. Heme also gives myoglobin (and meat) its red color.
Heme consists of a tetrapyrrole ring structure called protoporphyrin IX. Explore the structures, then select the correct terms to fill in the blanks. If you are not a biochemist, those terms probably sound like a foreign language! Look at the structures below. See if you can identify the pyrole ring and porphyrin within the protoporphyrin IX molecule.
pyrrole ringA _____ is a five-membered closed ring containing four carbon and one nitrogen atoms. Four of these rings are arranged in a square planar structure and joined together by =CH- groups to form a ________. In this structure, all the nitrogen atoms point to the center of the structure. [Word bank: pyrrole ring; porphyrin; protoporphyrin IX; heme group; magnesium; iron; hydrogen]
Myoglobin can be found in two states - either without oxygen (deoxymyoglobin) or bound to oxygen (oxymyoglobin). Click the buttons below to explore the structure of these two forms of myoglobin in sperm whale. Note that for crystallization purposes, the iron (Fe) atom in the center of the heme was substituted with cobalt (Co).
oxy myoglobin from sperm whale; PDB ID 1YOIOxygen is transported in the blood by hemoglobin, which is a member of the globin family. Like myoglobin, peptide chains contain only helices, and each peptide chain contains a single heme group. Unlike myoglobin, hemoglobin has quaternary structure. It consists of two α-globin chains and two β-globin chains. First we'll explore the structure of the β-globin chain, then we'll look at the whole molecule.
Deoxyhemoglobin has a low oxygen affinity. This means that it is not eager to pick up oxygen molecules. This is a good thing. Once hemoglobin has transported oxygen throughout the body, it wouldn't be good if it picked up the oxygen again and took it back to the lungs. Some textbooks refer to deoxyhemoglobin as the T form of hemoglobin. This refers to one of two structural conformations of hemoglobin.
When deoxyhemoglobin arrives in the lungs, there is a high concentration of oxygen - so high that the deoxyhemoglobin is able to pick up an oxygen molecule. When one oxygen molecule binds to hemoglobin, a conformational change occurs in hemoglobin, converting it to the R form. The R form has a much higher oxygen affinity. So the molecule quickly picks up four oxygen molecules (one for each heme group in each hemoglobin subunit).
Oxyhemoglobin is transported in the blood throughout the body, where the oxygen concentration is much lower. Oxygen is more likely to detach from the heme group in this environment. Once one oxygen molecule is released, hemoglobin undergoes a second conformational change, from the R state back to the T state. Since hemoglobin in the T state doesn't hold oxygen well, the oxygen is quickly released into the low oxygen environment in the body. Deoxyhemoglobin is then transported through the blood back to the lungs, and the process begins again.
Let's look at the shape of the heme group in the β-globin subunit of hemoglobin in the absence and presence of oxygen.
In deoxyhemoglobin, the heme group isn't perfectly flat. It is slightly dome-shaped, and the central iron atom (orange) lies slightly outside the plane of the heme group, towards the histidine residue. Look at the structure below and find the iron atom.
human deoxy hemoglobin heme group; PDB ID: 2HHBIn oxyhemoglobin, the oxygen binds opposite the histidine residue, and it 'tugs' at the iron atom, pulling it into the plane of the heme group. This change in the position of the iron atom has a domino effect on the whole protein, resulting in the conformational change between R and T. Locate the iron atom (orange) and bound oxygen (double red spheres) in the structure below.
human oxy hemoglobin - beta chain; PDB ID: 1HHOThe conformational change in hemoglobin involves a second molecule, BPG (2,3-bisphosphoglycerate), which binds to the T conformation (deoxyhemoglobin) and stabilizes it. But BPG can't bind to the R conformation (oxyghemoglobin). The conformational change can most easily be seen by looking at the distance between residues lys40 in the A chains and his146 in the B chains. In the images below, these distance between these two residues is indicated with a dotted black line; the number indicates the distance between the two selected atoms.
deoxyhemoglobin with BPG; PDB ID 1B86