Protein crystallization
Encyclopedia
Most Proteins and many biological macromolecules differ from "small" molecules because the environment in which they function is aqueous. Therefore most biological macromolecules can be prompted to form crystals when the solution in which they are dissolved becomes supersaturated. The manner in which this occurs is typical of many other compounds that crystallize from solution
. Membrane proteins are crystallized after being solubilized in detergent
solutions and certain seed
proteins like crambin
that is not soluble in water, but in ethanol are crystallized by adding water instead. . Apart, from the difference in solvent, standard protein crystallization and seeding techniques like streak seeding
are still used in protein crystallization from organic solvent. Like for other compounds, protein crystallization occurs more readily when the protein has been purified and is homogeneous. Most proteins structures currently available are from proteins that have been crystallized from aqueous solutions. When forming a crystal individual protein molecules align themselves in a repeating series of unit cells by adopting a consistent orientation. The crystalline lattice that forms is held together by noncovalent interactions . The importance of protein crystallization is that it serves as the basis for X-ray crystallography
, wherein a crystallized protein is used to determine the protein’s three-dimensional structure via X-ray diffraction. In 1934, John Desmond Bernal and his student Dorothy Hodgkin discovered that protein crystals surrounded by their mother liquor gave better diffraction patterns than dried crystals. Using pepsin
, they were the first to discern the diffraction pattern of a wet, globular protein. Prior to Bernal and Hodgkin, protein crystallography had only been performed in dry conditions with inconsistent and unreliable results .
For a better part of the 20th century, progress in determining protein structure was slow due to the difficulty inherent in crystallizing proteins. When the Protein Data Bank
was founded in 1971, it contained only seven structures. Since then, the pace at which protein structures are being discovered has grown exponentially, with the PDB surpassing 20,000 structures in 2003, and containing over 68,000 as of 2010.
. Protein crystallization is inherently difficult because of the fragile nature of protein crystals. Proteins have irregularly shaped surfaces, which results in the formation of large channels within any protein crystal. Therefore, the noncovalent bonds that hold together the lattice must often be formed through several layers of solvent
molecules .
In addition to overcoming the inherent fragility of protein crystals, a number of environmental factors must also be overcome. Due to the molecular variations between individual proteins, conditions unique to each protein must be obtained for a successful crystallization. Therefore, attempting to crystallize a protein without a proven protocol can be very challenging and time consuming.
, concentration of protein, temperature, and precipitants. The more homogenous a protein in solution, the better the chances are for it to form a crystal. Typical standards have the protein solution being at least 97% pure. pH conditions are very important due to the fact that different pHs can result in different packing orientations. Buffers, such as Tris-HCl, are often necessary for the maintenance of a particular pH . Precipitants, such as ammonium sulfate
or polyethylene glycol
, are compounds that cause the protein to precipitate out of solution .
Simply put, the hanging drop method differs from the sitting drop method in the vertical orientation of the protein solution drop within the system. It is important to mention that both methods require a closed system, that is, the system must be sealed off from the outside using an airtight container or high-vacuum grease between glass surfaces. The images to the right depict the hanging drop and sitting drop systems .
Robots can be used to set up and automate large number of crystallization experiments simultaneously. What would otherwise be slow and potentially error-prone process carried out by a human can be accomplished efficiently and accurately with an automated system. Robotic crystallization systems use the same components described above, but carry out each step of the procedure quickly and with a large number of replicates. Each experiment utilizes tiny amounts of solution, and the advantage of the smaller size is two-fold: the smaller sample sizes not only cut-down on expenditure of purified protein, but smaller amounts of solution lead to quicker crystallizations. Each experiment is monitored by a camera which detects crystal growth .
, also known as protein NMR, which is best suited to small proteins, or transmission electron microscopy
, which is best suited to large proteins or protein complexes.
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
. Membrane proteins are crystallized after being solubilized in detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
solutions and certain seed
Seed
A seed is a small embryonic plant enclosed in a covering called the seed coat, usually with some stored food. It is the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant...
proteins like crambin
Crambin
Crambin is a small seed storage protein from the Abyssinian cabbage. It has 46 residues .It has been extensively studied by X-ray crystallography since its crystals are unique and diffract to of 0.48 Å resolution....
that is not soluble in water, but in ethanol are crystallized by adding water instead. . Apart, from the difference in solvent, standard protein crystallization and seeding techniques like streak seeding
Streak seeding
Streak seeding is a method first described by Enrico Stura to induce crystallization in a straight line into a sitting or hanging drop for protein crystallization. The purpose is to control nucleation and understand the parameters that make crystals grow...
are still used in protein crystallization from organic solvent. Like for other compounds, protein crystallization occurs more readily when the protein has been purified and is homogeneous. Most proteins structures currently available are from proteins that have been crystallized from aqueous solutions. When forming a crystal individual protein molecules align themselves in a repeating series of unit cells by adopting a consistent orientation. The crystalline lattice that forms is held together by noncovalent interactions . The importance of protein crystallization is that it serves as the basis for X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
, wherein a crystallized protein is used to determine the protein’s three-dimensional structure via X-ray diffraction. In 1934, John Desmond Bernal and his student Dorothy Hodgkin discovered that protein crystals surrounded by their mother liquor gave better diffraction patterns than dried crystals. Using pepsin
Pepsin
Pepsin is an enzyme whose precursor form is released by the chief cells in the stomach and that degrades food proteins into peptides. It was discovered in 1836 by Theodor Schwann who also coined its name from the Greek word pepsis, meaning digestion...
, they were the first to discern the diffraction pattern of a wet, globular protein. Prior to Bernal and Hodgkin, protein crystallography had only been performed in dry conditions with inconsistent and unreliable results .
For a better part of the 20th century, progress in determining protein structure was slow due to the difficulty inherent in crystallizing proteins. When the Protein Data Bank
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....
was founded in 1971, it contained only seven structures. Since then, the pace at which protein structures are being discovered has grown exponentially, with the PDB surpassing 20,000 structures in 2003, and containing over 68,000 as of 2010.
Overview
The goal of crystallization is to produce a well-ordered crystal that is lacking in contaminants while still large enough to provide a diffraction pattern when exposed to X-rays. This diffraction pattern can then be analyzed to discern the protein’s tertiary structureTertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
. Protein crystallization is inherently difficult because of the fragile nature of protein crystals. Proteins have irregularly shaped surfaces, which results in the formation of large channels within any protein crystal. Therefore, the noncovalent bonds that hold together the lattice must often be formed through several layers of solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
molecules .
In addition to overcoming the inherent fragility of protein crystals, a number of environmental factors must also be overcome. Due to the molecular variations between individual proteins, conditions unique to each protein must be obtained for a successful crystallization. Therefore, attempting to crystallize a protein without a proven protocol can be very challenging and time consuming.
Conditions
Several conditions come into factor if a protein sample will crystallize or not. Some of these factors include protein purity, pHPH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
, concentration of protein, temperature, and precipitants. The more homogenous a protein in solution, the better the chances are for it to form a crystal. Typical standards have the protein solution being at least 97% pure. pH conditions are very important due to the fact that different pHs can result in different packing orientations. Buffers, such as Tris-HCl, are often necessary for the maintenance of a particular pH . Precipitants, such as ammonium sulfate
Ammonium sulfate
Ammonium sulfate , 2SO4, is an inorganic salt with a number of commercial uses. The most common use is as a soil fertilizer. It contains 21% nitrogen as ammonium cations, and 24% sulfur as sulfate anions...
or polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
, are compounds that cause the protein to precipitate out of solution .
Vapor Diffusion
Two of the most commonly used methods for protein crystallization fall under the category of vapor diffusion. These are known as the hanging drop and sitting drop methods. Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete .Simply put, the hanging drop method differs from the sitting drop method in the vertical orientation of the protein solution drop within the system. It is important to mention that both methods require a closed system, that is, the system must be sealed off from the outside using an airtight container or high-vacuum grease between glass surfaces. The images to the right depict the hanging drop and sitting drop systems .
High Through-Put Methods
High through-put methods exist to help streamline the large number of experiments required to explore the various conditions that are necessary for successful crystal growth. There are numerous commercials kits available for order which apply preassembled ingredients in systems guaranteed to produce successful crystallization. Using such a kit, a scientist avoids the hassle of purifying a protein and determining the appropriate crystallization conditions.Robots can be used to set up and automate large number of crystallization experiments simultaneously. What would otherwise be slow and potentially error-prone process carried out by a human can be accomplished efficiently and accurately with an automated system. Robotic crystallization systems use the same components described above, but carry out each step of the procedure quickly and with a large number of replicates. Each experiment utilizes tiny amounts of solution, and the advantage of the smaller size is two-fold: the smaller sample sizes not only cut-down on expenditure of purified protein, but smaller amounts of solution lead to quicker crystallizations. Each experiment is monitored by a camera which detects crystal growth .
Alternatives
Some proteins do not fold properly outside their native environment, e.g. proteins which are part of the cell membrane like ion channels and G-protein coupled receptors, their structure is altered by interacting proteins or switch between different states. All those conditions prevent crystal growth or give crystal structures which do not represent the natural structure of the protein. In order to determine the 3D structure of proteins which are hard to crystallize researchers may use nuclear magnetic resonanceNuclear magnetic resonance
Nuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
, also known as protein NMR, which is best suited to small proteins, or transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
, which is best suited to large proteins or protein complexes.
External links
- "Protein Crystallization and Dumb Luck". An essay on the haphazard side of protein crystallization by Bob Cudney: http://www.msc.com/downloads/journal/Vol16.2.1999/cudney.pdf
See also
- Crystal engineeringCrystal engineeringCrystal engineering is the design and synthesis of molecular solid-state structures with desired properties, based on an understanding and exploitation of intermolecular interactions. The two main strategies currently in use for crystal engineering are based on hydrogen bonding and coordination...
- Crystal growthCrystal growthA crystal is a solid material whose constituent atoms, molecules, or ions are arranged in an orderly repeating pattern extending in all three spatial dimensions. Crystal growth is a major stage of a crystallization process, and consists in the addition of new atoms, ions, or polymer strings into...
- Crystal opticsCrystal opticsCrystal optics is the branch of optics that describes the behaviour of light in anisotropic media, that is, media in which light behaves differently depending on which direction the light is propagating. The index of refraction depends on both composition and crystal structure and can be...
- Crystal systemCrystal systemIn crystallography, the terms crystal system, crystal family, and lattice system each refer to one of several classes of space groups, lattices, point groups, or crystals...
- Crystallization processes
- Crystallographic databaseCrystallographic databaseA crystallographic database is a database specifically designed to store information about crystals and crystal structures. Crystals are solids having, in all three dimensions of space, a regularly repeating arrangement of atoms, ions, or molecules. They are characterized by symmetry, morphology,...
- Crystallographic group
- DiffractionDiffractionDiffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...
- Electron crystallographyElectron crystallographyElectron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope .- Comparison with X-ray crystallography :...
- Electron diffractionElectron diffractionElectron diffraction refers to the wave nature of electrons. However, from a technical or practical point of view, it may be regarded as a technique used to study matter by firing electrons at a sample and observing the resulting interference pattern...
- Neutron crystallography
- Neutron diffractionNeutron diffractionNeutron diffraction or elastic neutron scattering is the application of neutron scattering to the determination of the atomic and/or magnetic structure of a material: A sample to be examined is placed in a beam of thermal or cold neutrons to obtain a diffraction pattern that provides information of...
- X-ray crystallographyX-ray crystallographyX-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
- X-ray diffraction