Time resolved crystallography
Encyclopedia
Time resolved crystallography utilizes X-ray crystallography
imaging to visualize reactions in four dimensions (x, y, z and time). This enables the studies of dynamical changes that occur in for example enzymes during their catalysis. The time dimension is incorporated by triggering the reaction of interest in the crystal prior to X-ray
exposure, and then collecting the diffraction patterns at different time delays. In order to study these dynamical properties of macromolecule
s three criteria must be met;
This has led to the development of several techniques that can be divided into two groups, the pump-probe method and diffusion-trapping methods.
pattern is collected by an X-ray pulse (probe) at a specific time delay. This makes it possible to obtain many images at different time delays after reaction triggering, and thereby building up a chronological series of images describing the events during reaction.
To obtain a reasonable signal to noise ratio this pump-probe cycle has to be performed many times for each spatial rotation of the crystal, and many times for the same time delay. Therefore the reaction that one wishes to study with pump-probe must be able to relax back to its original conformation after triggering, enabling many measurements on the same sample.
, use of inhibitor or lowering the temperature in order to slow down the turnover rate or maybe even stop the reaction completely at a specific step. Just starting the reaction and then flash-freeze it, thereby quenching it at a specific time step, is also a possible method. One drawback with diffusion-trapping methods is that they can only be used to study intermediates that can be trapped, thereby limiting the time resolution one can obtain through the methods as compared to the pump-probe method.
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...
imaging to visualize reactions in four dimensions (x, y, z and time). This enables the studies of dynamical changes that occur in for example enzymes during their catalysis. The time dimension is incorporated by triggering the reaction of interest in the crystal prior to X-ray
X-ray
X-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...
exposure, and then collecting the diffraction patterns at different time delays. In order to study these dynamical properties of macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
s three criteria must be met;
- The macromolecule must be biologically active in the crystalline state
- It must be possible to trigger the reaction in the crystal
- The intermediate of interest must be detectable, i.e. it must have a reasonable amount of concentration in the crystal (preferably over 25%).
This has led to the development of several techniques that can be divided into two groups, the pump-probe method and diffusion-trapping methods.
Pump-probe
In the pump-probe method the reaction is first triggered (pump) by photolysis (most often laser light) and then a diffractionDiffraction
Diffraction 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...
pattern is collected by an X-ray pulse (probe) at a specific time delay. This makes it possible to obtain many images at different time delays after reaction triggering, and thereby building up a chronological series of images describing the events during reaction.
To obtain a reasonable signal to noise ratio this pump-probe cycle has to be performed many times for each spatial rotation of the crystal, and many times for the same time delay. Therefore the reaction that one wishes to study with pump-probe must be able to relax back to its original conformation after triggering, enabling many measurements on the same sample.
Diffusion-trapping
Diffusion-trapping methods utilizes diffusion techniques to get the substrates into the crystal and thereafter different trapping techniques are applied to get the intermediate of interest to accumulate in the crystal prior to collection of the diffraction pattern. These trapping methods could involve changes in 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...
, use of inhibitor or lowering the temperature in order to slow down the turnover rate or maybe even stop the reaction completely at a specific step. Just starting the reaction and then flash-freeze it, thereby quenching it at a specific time step, is also a possible method. One drawback with diffusion-trapping methods is that they can only be used to study intermediates that can be trapped, thereby limiting the time resolution one can obtain through the methods as compared to the pump-probe method.