Structural Biochemistry, Proteins, Neutron Diffraction

Protein crystal , (PCG) Protein Crystal Growth Porcine Elastase. This enzyme is associated with the degradation of lung tissue in people suffering from emphysema. It is useful in studying causes of this disease. (Image: Public domain)

Protein crystal , Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason, the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. (Image: Public domain)

Neutron Diffraction (also known as neutron scattering or neutron crystallography) is an experimental science that studies the spatial arrangement of atoms in proteins. Although neutron diffraction and X-ray scattering techniques use different radiation sources, the resulting diffraction pattern is analyzed using the same coherent imaging techniques. However, the use of neutron diffraction as an experimental technique is still a relatively new technique compared to X-ray and electron diffraction because the accumulation of free neutrons, the radiation source, can only be obtained from nuclear reactors.

Although neutron diffraction has been used as an experimental technique in physics since the early 1900s, its application in chemistry and biology did not start until the 1980s. In 1984, Wlodawer, Walter, Huber, and Sjolin collaborated to bring X-ray crystallography and neutron diffraction methods together to come up with novel methods of determining internal dynamics of protein molecules. Their experiment utilized the joint application of two methods to determine the structure of a new crystal form of bovine pancreatic trypsin inhibitor (BPTI). The project is the first to reveal the atomic position of proteins to a size within 0.1 nanometers (i.e. the diameter of a hydrogen atom), and it is the first detailed analysis of how protein structure is affected by molecular packing.

In 1994, Clifford G. Shull was awarded the Nobel Prize in Physics for developing
a new way to use neutron diffraction. From this point on,
neutron diffraction took new life with the new usage of scattering, a technique that
allowed scientists to observe that the dynamics of atom movement and excitation.
He found that when a material was shot with a beam of neutrons, the neutrons bounced
off of atoms and hit other atoms thus scattering light and making neutrons go in every which
way and direction. This created a general pattern that we can use to deduce the surrounding
substituents and atom placement.

By understanding these substituents and their placements we can develop more insight into the intrinsic nature
of molecules and atoms. According to Clifford, these new discoveries would pave the way for "better semiconductors, better microphones, better window glass, etc."
Shull worked as a professor at MIT for many years. When he won the Nobel prize award in 1994, he was already retired from
teaching but continued to conduct research. Ever since his innovative technique of
neutron scattering, he was hailed as the father of neutron scattering.

Fundamentally neutron diffraction relies upon the fact that free neutrons exhibit wave-like diffraction behaviors, and this happens when the radiation wave encounters obstacles (e.g. proteins) that is comparable with the wavelength. Generally, the effects of diffraction are more pronounced for waves where the wavelength is on the order of the size of the diffracting object. Neutron diffraction is a type of elastic scattering, meaning that the incoming energy of the neutron is equivalent to the outgoing energy after scattering occurs.

Additionally, the method is a unique tool for studying a wide variety of materials with magnetic properties because neutrons have magnetic moments that can interact with orbital and spin moments in magnetic atoms.

To study crystalline solids and molecules and their structure it is useful to free these neutrons and excite them the intensity pattern and formation of the excitation gives us information about the structure of a molecule. These neutrons are not found in nature at least
not for a very long period of time. Nuclear reactors can set these neutrons free
and we can study diffraction of these neutrons. By studying their wavelength and quantum properties we essentially create a sample, not unlike
x-ray diffraction. Neutron diffraction is similar and generates structural information much like electron diffraction but
neutron beams actually have a strong affinity to react with the internal nuclei of cells than do x-rays. Also when studying neutrons we have more insight into the positioning of protons. X-rays at most times can destroy
or denature a material under study due to their intensive X-rays in which case neutron diffraction can be very beneficial.

The scattering effect of the neutrons can be explained through two different phenomena. Firstly there is a
close proximity reaction between the neutron and the atomic nucleus because these neutrons have a natural affinity to do so.
This interaction is specific to each atomic number because the atomic nucleus is classified as a point scatterer, thus producing
isotropic scattering. The second interaction relates to the function of magnetism and spin. The magnetic moment of the neutron
is directly connected with the spin and orbital hybridization and arrangement of the molecule or atom. Detailed data can be obtained through this
magnetic approach study which is absent in other forms of crystallography.

With the knowledge that scattering patterns do not vary from atom to atom with the same atomic number, we can substitute different enriched isotopes to get a
more panoramic and holistic study of the molecule. Because neutrons are not charged, we do not have to worry
about the possibility of them interacting heavily with the electron cloud surrounding the atom. This solves the problem
of electron diffraction. Neutrons will only directly react with the nucleus.

How does neutron diffraction help in studying crystallized lattice structures? Inelastic neutron scattering studies
vibrational thermodynamics which elucidates the equilibrium structure. Lattice vibration can be induced with neutrons
that are low in energy, they can also be induced to release ponons or quanta. With this diffraction pattern, we can relate vibrational modes of each part of the crystal and calculate dispersion relations and then construct an idea
of its structure.

(Image: Pkleong at English Wikibooks, Public domain)

The essence of neutron diffraction, as noted by its "father," Clifford Shull, was to identify hydrogen atoms
and how they appeared to go about processes in biological materials or inorganic substances. Hydrogen containing
structures are very much present in our world and necessary for the development of drugs, resources, and other endeavors.
With the use of neutrons, we can exploit nucleus relations with protons and neutrons without being
disturbed by the electron cloud.

Neutron Diffraction can be used to determine the atomic structure of low atomic number molecules such as proteins because low atomic number materials have higher nucleus cross-sectional areas for neutrons to interact with. This method of crystallographic experiment is similar to X-ray diffraction; however, the fact that neutrons are scattered by the nucleus rather than electrons in an atom means that the effect of diffraction is independent of atomic number. Furthermore, this diffraction method works because neutrons have energies with equivalent wavelengths in the 0.1 nanometers and are therefore suitable for interatomic interference studies.

Using neutron diffraction to determine protein structures requires several steps. First, it requires careful preparation of the protein crystals, for without perfect crystals of protein it is impossible to carry out any crystallographic structural studies. The aim of protein crystallization is to produce well-ordered crystals that are large enough to diffract neutron beam. Therefore, the crystallization process is long and is often the rate-limiting step in the experiment. After the derivative protein crystals become available, the crystals are mounted and neutron diffraction images are taken.

Neutron diffraction in the industrial world has been used to probe structures and magnetism of condensed matter. Industrial interests such as mechanical behavior in materials.
Engineers must study strain on certain molecules to understand strain mapping. Of course, all
mechanical behavior starts with microscopic scales of structural features. Engineers must take into account
residual stress which can be studied through neutron diffraction. When applying neutron diffraction to engineering work,
it is referred to as engineering diffraction.

Neutron diffraction is very sensitive to small nuances in structure thus creating a multitude
of different peaks that depend on three factors. These three factors are peak position, peak width, and integrated intensity.
This allows engineers to access the texture, strain, and strain fluctuation of a sample. The principle of understanding
strain studies is based on Bragg's law.

So what kind of engineering situations would call for neutron diffraction? One practical application is that of welding. When welds have nonuniform expansion or shrinkage in heat directed zones during times of intense heat when the metal alloys as passed through a weld pass,
residual stress is observed. Residual stress can include cracking and change in shape. This could limit the quality of a product
and even corrode machinery. Neutron diffraction provides a 3-D spatial
distribution of the tensor of stress. This can be used in all kinds of weld
materials. Different materials must take into account different parameters and geometries. In short, engineers
depend on the uniformity, rigidity, and strength of their materials and when these things fail, scientific approaches
such as neutron diffraction can help identify the problem and innovate new ideas of the solution to create and facilitate
advances in engineering materials. Thus neutron diffraction is a precursor to all industrial and practice clauses.

Neutron Peaks and Diffraction , Applications in Industry and Engineering (Image: Pkleong at English Wikibooks, Public domain)

• Single Crystal Neutron Diffraction and Protein Crystallography
• the need for new spectroscopic methods of determining protein structures
• Neutron Diffraction

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