Published on September 18th, 2012 | by Leidamarie Tirado-Lee0
How It’s Made: Protein Structures by X-ray Crystallography
If DNA is the blueprint that guides the construction of an organism, then proteins are the workhorses that allow for the organism to function. Need to catalyze a reaction? There are proteins for that! Need to provide structure or support? There are proteins for that! Given the importance of proteins for life, understanding exactly how proteins do those crazy things they do is a big focus of biophysical research.
Just as a building’s design greatly dictates its utility so too does protein structure influence its functionality. Solving protein structures is therefore a highly attractive endeavor for advancing our understanding of diseases and for developing novel therapeutics.
A Brief Discussion of Protein Structure
Proteins are polymers of covalently bonded amino acid building blocks. There are 20 amino acids that can come together to form chains, which can range from a few units (dipeptides, tripeptides) to thousands of units (polypeptide). When it comes to function, size doesn’t necessarily matter! Many important hormones are peptide chains only a few residues in length. Did you know that aspartame, a common artificial sweetener, is a peptide three amino acids long?
The structure of a chain of amino acids can be described at a number of different levels. (see figure below). Primary structure, the most basic level, is simply a linear representation of amino acid sequence. Secondary structure refers to the local, stable patterns (alpha helices, beta sheets, etc.) often adopted by strings of amino acids. The distinct three-dimensional structure or tertiary structure is created by the sum of all of the folds in the protein. Lastly, quaternary structure describes how multiple folded peptides can come together in a large complex.
X-ray Crystallography: A window to the protein’s soul
They say “nothing worth having comes easily” and protein structure determination is a great example. For one, proteins are tiny. Even the largest proteins only measure several nanometers. To put that into perspective, trying to view a protein is like trying to make out details on the Earth’s surface from the moon! Therefore, really powerful tools must be used to “see” a protein structure.
Enter X-ray crystallography, which has determined the most protein structure models to date. The basic set-up (shown on right) seems relatively straightforward even if the individual steps are not so in practice. First you need to get a protein crystal. Getting these protein crystals is by far one of the hardest steps and I plan to devote a whole entry to this alone (try to contain your excitement). Next, subject the protein crystal to an x-ray beam. Since x-rays are electromagnetic waves like visible light, when then x-ray beam hits the crystal the x-rays will scatter.
But what is it that we are really seeing? By growing protein crystals, one is able to obtain many ordered protein molecules in identical orientations. This is key because individual molecules would diffract too weakly to detect. This is true in the same way that it’s harder to see one grain of sand than a beach. Thus having the high concentration of ordered molecules helps to produce a strong diffraction for structure determination.
Now, it is actually the electron clouds within these molecules that scatter the x-ray beam. The scattered x-rays are captured by a detector to obtain the diffraction pattern, which consists of a series of spots, each corresponding to a diffracted beam, whose directions and intensities are used by a computer program to model the structure of the crystallized protein. Therefore, the diffraction pattern provides information about how the electrons in the molecule are distributed, commonly referred to as the electron density. It is this electron density that describes the shape of the protein molecule as interpreted by the x-ray crystallography experiment. As a final step, the crystallographer uses the density map to develop a model of the protein.
If you are interested in learning more about crystallography I recommend Crystallography Made Crystal Clear by Gale Rhodes.
Feature image by Argonne National Laboratory.