Seeing the really, really small
When we use a light microscope to examine pond water we enter into an exciting world, which is full of all sorts of things that our naked eyes cannot see. There really is something magical about watching a paramecium swimming or an amoeba crawling in pond scum. But microscopes have their limitations. At some point, things become so small that we cannot observe them even in the best of microscopes. Protein molecules, for example, are simply too small to be seen underneath a microscope.
Measurement and proteins
Chemists and biochemists often use a unit of measure called an ångström. It is abbreviated as Å. One angstrom is equal to 0.0000000001 meters (or 1.0 x 10-10 meters). In other words, an ångström represents a very small number. The height of a typical first grader is ~10,000,000,000 Å. We don’t need a microscope to see that pesky first grader. The diameter of a cell from your body is ~500,000 Å, and we can see it under a light microscope. The diameter of a bacterium is ~50,000 Å so if we are using a good microscope, we might be able to see it. But proteins have diameters of ~50 Å, so it is not possible to observe them directly in a microscope because they are simply too small!
X-ray Crystallographic method
How then do we know what proteins or sugars or fats look like in three-dimensions? We know by using a technique called X-ray crystallography. It is a very sophisticated method that uses crystals, X-rays, and lots of computing power. The first step is to grow crystals of the molecule that we are interested in. There are lots of ways to produce crystals. The next step is to put the crystals in a special X-ray beam. The crystal scatters the X-rays onto an electronic detector, which functions as a recorder. With specialized computer programs (or in today’s jargon “apps”), it is possible to use the information gathered on the detector to construct a so-called “electron density map,” which is basically a roadmap that tells us what the molecule looks like in three-dimensions. From the map, a model of the molecule is constructed using specialized computer graphics programs. The process is summarized in the figure below.
History of X-ray crystallography
X-ray crystallography has a long and illustrious history. In 1895 Wilhelm Röntgen, a German physicist, discovered X-rays and demonstrated that this type of radiation could pass directly through human tissues leaving behind images of bones. He won the Nobel Prize in Physics in 1901. Another German physicist, Max von Laue proved in 1912 that crystals could diffract X-rays. He later went on to win the Nobel Prize in Physics in 1914. The first X-ray structure solved was that of table salt in 1913. A father and son team, William Henry Bragg and William Lawrence Bragg, respectively, made additional and substantial contributions to the field of X-ray crystallography, and together they shared the 1915 Nobel Prize for their pioneering efforts. The 1946 Nobel Prize in Chemistry was awarded to James B. Sumner who discovered that proteins could be crystallized. The first protein structures ever solved were myoglobin and hemoglobin. These are proteins that carry oxygen in your body. The work was carried out by John Kendrew and Max Perutz, and together they shared the Nobel Prize in 1962 in Chemistry.
In the same year, Francis Crick, John Watson, and Maurice Wilkins received the Nobel Prize in Physiology/Medicine for their pioneering contributions towards the understanding of DNA, the molecule that carries your genetic information. Interestingly, it was Rosalind Franklin who generated the X-ray diffraction patterns that led to the discovery of the DNA double helix. Unfortunately her contributions to the field were ignored by the Nobel Prize committee, even though it was her data that Crick, Watson, and Wilkins used. Science has its politics, unfortunately.
X-ray crystallographers have continued to receive Nobel Prizes to date, and it is not possible to list them all. But some of the more noteworthy winners include Dorothy Hodgkin who won it in 1964 for solving the structures of important substances such as Vitamin B12, Johan Deisenhofer, Robert Huber, and Hartmut Michel who won it in 1988 for solving the structure of the first membrane-bound protein, and Venkatraman Ramakrishnan, Thomas Steitz, and Ada Yonath who won it in 2009 for determining the structure of the ribosome, a cellular organelle that is responsible for building proteins in your body.Perhaps the most amazing thing about the history of X-ray crystallography is that it took Max Perutz and John Kendrew nearly 20 years to solve the structures of myoglobin and hemoglobin. Yet, with today’s computing power and intense X-ray beams, once good crystals are obtained it can take a day to solve the structure if all the experiments go well. The field has, indeed, witnessed a technical revolution over the last 50 years.
Importance of X-ray crystallography
One question we might ask is why knowing the structure of a protein is important. Well, here is an example. Suppose there is a protein in your body that is responsible for maintaining proper blood pressure, and it isn’t functioning correctly so your blood pressure rises. By knowing the structure of the protein in three-dimensions it is possible to design a drug that will stop the protein from “over-working” so that your blood pressure goes down. This process of determining the structure of a protein and designing molecules that will bind to it to affect its function is known as “structure-based” drug design. Pharmaceutical companies use this very technique for making new drugs to treat all sorts of things such as high blood pressure or high cholesterol. And this technique played a key role in the development of inhibitors to fight the human immunodeficiency virus (HIV), which is the causative agent of acquired immunodeficiency syndrome or AIDS.