Beat Blattmann and Patrick Sticher from the University of Zürich, Switzerland, explain the science behind protein crystallography and provide a protocol for growing your own crystals from protein – an essential method used by scientists to determine protein structures.
In 1959, Max Perutz and John Kendrew published an article on the three-dimensional structure of whale myoglobin, which is a small protein responsible for the transport of oxygen in whale cells. By investigating the protein’s structure, the two scientists wanted to understand the oxygen-carrying mechanism at the molecular level. They grew crystals from this protein and managed to determine its structure by analysing the X-ray diffraction pattern of the crystal.
A number of myoglobins from other species had been tested before with little success, until Perutz and Kendrew obtained a useable diffraction pattern with whale myoglobin crystals. This pioneering work was awarded the Nobel Prize in Chemistry in 1962w1. Fifty years later, however, it is still a challenge to obtain protein crystals for structural studies.
Proteins are the largest group of non-aqueous components in living cells. Almost every biochemical reaction requires a specific protein, called an enzyme. Other types of proteins have mechanical and structural functions (e.g. collagen in connective tissue), or mediate cell signalling (e.g. hormone receptors), immune responses (e.g. antibodies) or the transport of small molecules (e.g. ion channels). The variety is immense: more than 20 000 different proteins are known to exist in humans alone.
Despite this variety, all proteins share an identical structural principle. They consist of 20 different building blocks, called amino acids, which are arranged in a linear chain connected by covalent bonds between adjacent amino acids (see figure below). The length of the protein chain varies from a few dozen to thousands of amino acids. In cells, each protein is assembled using the information encoded in its corresponding gene. The assembly is performed by a ribosome, which is a complex molecular machinery consisting of proteins and RNA.
Under natural conditions, the linear chains of amino acids spontaneously fold into distinct three-dimensional structures. Stretches of amino acids form typical secondary structural elements. The most prominent elements are ?-helices and ?-sheets (see figure below), which are typically stabilised by hydrogen bonds between individual amino-acid residues. The entire protein forms a tertiary structure consisting of a variety of such structure elements.
a. Proteins are built from amino acids, which are covalently linked to form a linear chain
b. Proteins are folded to a three-dimensional structure that determines their function. Small stretches of the amino-acid chain form typical folds. Two prominent structural elements are α-helices and β-sheets.
The function of a particular protein depends on its three-dimensional structure. Only when the protein is folded, the specific amino acids of the protein are close enough to enable the formation of an active site. These sites can catalyse biochemical reactions, as in the case of enzymes, or form a specific binding site, as in the case of antibodies. Investigating the structural details of a protein is of great importance to understand how fundamental processes of life function at a molecular level: this is the research area of structural biologists. One of the major challenges in structural biology today is the elucidation of the structure, function and interaction of huge macromolecular complexes and membrane proteinsw2. Due to their complexity, these proteins are experimentally extremely challenging, and every time the structure of a protein is determined, it is a major achievement. Nevertheless, since they are involved in fundamental biological processes, there is a great interest in better understanding their structure and function, and scientists keep trying to crystallise them.
Proteins are tiny structures, measuring only a few nanometres (1 nm = 1 millionth of a mm). Particles that size cannot be observed even with the strongest light microscope, which has a maximum resolution of 1 micrometre (1 ?m = 1 thousandth of a mm). Three major technologies are available to make protein structures ‘visible’:
As more than 90% of all protein structures deposited in the publicly accessible protein database of biological macromoleculesw3 have been determined by X-ray diffraction, we will concentrate on this method. To learn more about the history of crystallography and the journey of a protein from lab to lab, until its structure is solved, see the article by Dominique Cornuéjols in this issue.
Crystallising proteins is a tricky task, because it is difficult to determine the right conditions under which each new protein will crystallise – sometimes, it even seems impossible. So to ensure reproducible crystal quality (i.e. that equally good crystals can be grown again), scientists use controlled experimental set-ups to crystallise their proteins. The most frequently used method in protein crystallography is the vapour diffusion method (seeimage): in this method, a small amount of a crystallisation solution is added to the reservoir of the crystallisation chamber. A drop of protein solution and a drop of the crystallisation solution are pipetted onto the sitting drop post that is located in the centre of this chamber.
Immediately after adding all solutions, the chamber is sealed to avoid evaporation. Since the concentration of salt ions is higher in the crystallisation solution than in the mixture on the sitting drop post, solvent molecules will move from the protein drop to the reservoir by vapour diffusion in the gas phase. During this process, the solubility of the protein in the drop decreases. The protein solution in the drop eventually becomes supersaturated, which is a thermodynamically unstable state. This causes some of the protein in the drop either to form crystal nuclei that finally grow into larger protein crystals (see image), or to precipitate as amorphous protein which is useless for X-ray analysis. Crystallisation and precipitation are competing processes, so it is extremely important to find the optimal conditions favouring crystallisation.
In this practical activity, students learn more about modern X-ray crystallography by determining the optimal crystallisation conditions for a protein. They investigate the formation of lysozyme crystals as a function of pH and salt concentration.
Lysozyme is a protein belonging to a family of anti-bacterial enzymes which damage bacterial cell walls. In humans, it is abundant in a number of secretions, such as tears, saliva and mucus. Large amounts of lysozyme can also be found in chicken egg whites.
The following aqueous stock solutions should be prepared in advance by the teacher:
|2.0 ml of 3M NaCl stock solution (end conc. 0.6 M)
7.0 ml DI-water
|3.0 ml of 3M NaCl stock solution (end conc. 0.9 M)
6.0 ml DI-water
|4.0 ml of 3M NaCl stock solution (end conc. 1.2 M)
5.0 ml DI-water
|5.0 ml of 3M NaCl stock solution (end conc. 1.5 M)
4.0 ml DI-water
|6.0 ml of 3M NaCl stock solution (end conc. 1.8 M)
3.0 ml DI-water
|7.0 ml of 3M NaCl stock solution (end conc. 2.1 M)
2.0 ml DI-water
|A||1.0 ml sodium citrate (end conc. 0.1 M), pH 3.5||A1||A2||A3||A4||A5||A6|
|B||B 1.0 ml sodium acetate (end conc. 0.1 M), pH 4.5||B1||B2||B3||B4||B5||B6|
|C||1.0 ml sodium acetate (end conc. 0.1 M), pH 5.5||C1||C2||C3||C4||C5||C6|
|D||1.0 ml sodium phosphate (end conc. 0.1 M), pH 6.5||D1||D2||D3||D4||D5||D6|
When your class has successfully grown protein crystals, please contact Dr Patrick Sticher at firstname.lastname@example.org. The Swiss NCCR (National Center of Competence in Research) Structural Biologyw2 has offered to produce an X-ray diffraction image for the first 10 school classes that successfully grow protein crystals using this protocol. X-ray measurements can be made either directly from school samples, or, if shipment is a problem, by reproducing the optimised crystallisation conditions found in your class and measuring those crystals. Together with the diffraction image, the scientists offer to send additional information on what they would do next with this information to obtain the actual structure, and a certificate if required.
Students can chat online with the scientists via Skypew4, after performing their own experiments. To make an appointment, email Patrick Sticher (email@example.com) to chat with him using the Skype account ‘proteincrystallography’.
A set of Powerpoint slides, images and further experiments are available onlinew5.
The following suppliersw6 provide the required materials and chemicals:
Here are some recommended protocols for growing non-protein crystals with younger students: