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Topic Name: ANL Researchers has Found Structure of Protein Collagen at Unprecedented Level of Detail Never Before Seen

Category: Genetic Engineering

Research persons: Joseph Orgel

Location: Argonne National Laboratory, U.S. Department of Energy, United States

Details

The structure and behavior of one of the most common proteins in our bodies has been resolved at a level of detail never before seen, thanks to new research performed at the Advanced Photon Source (APS) at the U.S. Department of Energy's Argonne National Laboratory.

Illinois Institute of Technology biologist Joseph Orgel used the high-energy X-rays produced by the APS to examine the structure of collagen, a protein that composes more than a quarter of all protein in the human body and forms the principal component of skin, teeth, ligaments, the heart, blood vessels, bones and cartilage. In these tissues, collagen molecules pack themselves into overlapping bundles called fibrils. These fibrils, which each contain billions of atoms, entwine themselves into collagen fibers that are visible to the naked eye.

Scientists have known the basic molecular structure of collagen since the 1950s, when several different international groups of scientists discovered that it had a triple-stranded helical structure. However, researches had never before had the ability to study the structure of an entire fibril in the same way that they could study an individual collagen molecule, according to Orgel.

Orgel and his team performed diffraction studies on intact collagen fibrils inside the tendons of rat tails in order to understand just how the protein functioned within unbroken tissue. "We tried to draw a highly accurate map of the molecular structure of tissues," Orgel said. "By doing so, we hope to transform a very basic understanding that we have of the molecular structure of tissue into a much more tangible form."

Since the scientists kept the tendon tissue intact, they could see how the collagen molecule binds to collagenases, a class of enzymes which when working properly help to regulate the normal growth and development of animals but when malfunctioning can lead to the metastasis of cancerous tumors or rheumatoid arthritis. The visualization of this interaction could help drug developers to create an inhibitor to prevent the pathological action of the enzyme, Orgel said.

Previous studies of the structure of collagen had looked only at crystals of small fragments of the protein, so scientists had little idea of how it looked within intact tissue. "It's impossible to get the information that we did by removing tiny chunks of the tissue," Orgel said. "We couldn't obtain this data by single-crystal crystallography. This research was made possible only because of the BioCAT beamline provided by the APS."

Note for High Energy X-rays
High Energy X-rays or HEX-rays are very hard X-rays, with 80 keV - 1000 keV typically one order of magnitude higher in energy than conventional X-rays. They are produced at modern synchrotron radiation sources such as the beamline ID15 at the ESRF. The main benefit is the deep penetration into matter which makes them a probe for bulk samples in physics and materials science and facilitates sample environment and operation in air. Scattering angles are small and diffraction directed forward allowing for simple detector setups.
High Energy X-Rays (HEX-rays) between 100 and 300 keV bear unique advantage over conventional hard X-rays, which lie in the range of 5-20 keV. They can be listed as follows:
High penetration into materials due to a strongly reduced photo absorption cross section. The photo-absorption strongly depends on the atomic number of the material and the X-ray energy. Several centimeter thick volumes can be accesses in steel and millimeters in lead containing samples.
The Ewald sphere has a ten times smaller curvature than in the low energy case and allows to map whole regions in a reciprocal lattice, similar to electron diffraction.
Access to diffuse scattering. This is absorption and not extinction limited at low energies while volume enhancement takes place at high energies. Complete 3D maps over several Brillouin zones can be easily obtained.
High momentum transfers are naturally accessible due to the high momentum of the incident wave. This is of particular importance for studies of liquid, amorphous and nanocrystalline materials as well as PDF-analysis.
Simple diffraction setups due to operation in air.
Diffraction in forward direction for easy registration with a 2D detector.
Negligible polarization effects due to relative small scattering angles.
Special non-resonant magnetic scattering.
LLL interferometry.
Access to high-energy spectroscopic levels, both electronic and nuclear.
Forward scattering and penetration make sample environments easy and straight forward.
Neutron-like, but complementary studies combined with high precision spatial resolution.
Cross sections for Compton scattering are similar to coherent scattering or absorption cross sections.
With this advantages, HEX-Rays can be applied for a wide range of investigations. An overview, which by far is not complete:
Structural investigations of real materials, such as metals, ceramics, liquids. In particular in-situ studies of phase transitions at elevated temperatures up to the melt of any metal. Phase transitions, recovery, chemical segregation, recrystallization, twinning and domain formation are a few aspects to follow in a single experiment.
Materials in chemical or operation environments, such as electrodes in batteries, fuel cells, high-temperature reactors, electrolytes etc. The penetration and a well-collimated pencil beam allows to focus in the region and material of interest while it undergoes a chemical reaction.
Study of 'thick' layers, such as oxidation of steel in its production and rolling process, which are too thick for classical reflectometry experiments. Interfaces and layers in complicated environments, such as the intermetallic reaction of ZINCALUME ® surface coating on industrial steel in the liquid bath.
In situ studies of industrial like strip casting processes for light metals. A casting setup can be set-up on a beamline and probed with the HEX-ray beam in real time.
Bulk studies in single crystals differ from studies in surface-near regions limited by the penetration of conventional X-rays. It has been found and confirmed in almost all studies, that critical scattering and correlation lengths are strongly affected by this effect.
Combination of neutron and HEX-ray investigations on the same sample, such as contrast variations due to the different scattering lengths.
Residual stress analysis in the bulk with unique spatial resolution in centimeter thick samples; in-situ under realistic load conditions.
In-situ studies of thermo-mechanical deformation processes such as forging, rolling, extrusion of metals.
Real time texture measurements in the bulk during a deformation, phase transition or annealing, such as in metal processing.
Structures and textures of geological samples which may contain heavy elements and are thick.
High resolution triple crystal diffraction for the investigation of single crystals with all the advantages of high penetration and studies from the bulk.
Compton spectroscopy for the investigation of momentum distribution of the valence electron shells.
Imaging and tomography with high energies. Dedicated sources can be strong enough to obtain 3D tomograms in a few seconds. Combination of imaging and diffraction is possible due to simple geometries. For example tomography combined with residual stress measurement or structural analysis.
No radiation damage of the sample, which can pin incommensurations or destroy the chemical compound to be analyzed.

Note for Collagen
Collagen is the main protein of connective tissue in animals and the most abundant protein in mammals, making up about 25% of the whole-body protein content.
Collagen is one of the long, fibrous structural proteins whose functions are quite different from those of globular proteins such as enzymes; tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside, but collagen is also found inside certain cells. Collagen has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and teeth. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form. It is also used in cosmetic surgery and burns surgery.
Collagen has been widely used in cosmetic surgery, as a healing aid for burn patients for reconstruction of bone and a wide variety of dental, orthopedic and surgical purposes. Some points of interest are:
when used cosmetically, there is a chance of allergic reactions causing prolonged redness; however, this can be virtually eliminated by simple and inconspicuous patch testing prior to cosmetic use, and
most medical collagen is derived from young beef cattle (bovine) from certified BSE (Bovine spongiform encephalopathy) free animals. Most manufacturers use donor animals from either "closed herds", or from countries which have never had a reported case of BSE such as Australia and New Zealand.
porcine (pig) tissue is also widely used for producing collagen sheet for a variety of surgical purposes.
due to the care in donor animal breeding and selection, as well as the technology used in the preparation of collagen from animal sources, the chance of immune reactions or disease transmission has been virtually eliminated.
alternatives using the patient's own fat, hyaluronic acid or polyacrylamide gel are readily available.
Collagens are widely employed in the construction of artificial skin substitutes used in the management of severe burns. These collagens may be derived from bovine, equine or porcine, and even human, sources and are sometimes used in combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances.
Collagen is also sold commercially as a joint mobility supplement. This lacks supportive research as the proteins would just be broken down into its base amino acids during digestion, and could go to a variety of places besides the joints depending upon need and DNA orders.
Recently an alternative to animal-derived collagen has become available. Although expensive, this human collagen, derived from donor cadavers, placentas and aborted fetuses, may minimize the possibility of immune reactions.

Note for Fibril
Fibril is a fine fiber approximately 1 nm in diameter.
Cytoplasmic fibrils are observed on the protoplasmic cylinders found in most spirochetal species, although no function of the cytoplasmic fibrils has been ascribed.
Polysaccharides, the union of several linked monosaccharides, sometimes serve as a structural compound. Cellulose, the most abundant organic compound on Earth, forms cable-like strings, known as fibrils in the tough walls that enclose plant cells. While cellulose is a compilation of glucose monomers, they form unbranched, long strands instead of coils like starch or glycogen. These are arranged in parallel lines which form on top of each other in an intricate layer through hydrogen bonding.

Note for Rheumatoid arthritis
Rheumatoid arthritis (RA) is traditionally considered a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and joint destruction. RA is a systemic disease, often affecting extra-articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles.
The name is derived from the Greek rheumatos means "flowing", and this initially gave rise to the term "rheumatic fever", an illness that can follow throat infections and which includes joint pain. The suffix -oid means "resembling", i.e. resembling rheumatic fever. Arthr means "joint" and the suffix -itis, a "condition involving inflammation". Thus rheumatoid arthritis was a form of joint inflammation that resembled rheumatic fever. Rheumatoid arthritis appears to have been described in paintings more than a century before the first detailed medical description of the condition in 1800 by Landre-Beauvais.
Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disorder affecting the joints and sometimes other organs as well. It is by definition polyarticular; that is, it affects many joints. Most commonly, the small joints in the hands and feet are affected, but larger joints (shoulders, knees etc) can also be affected; the pattern of joint involvement can differ from patient to patient.
Rheumatoid arthritis affects women three times more often than men, and it can first develop at any age. The risk of first developing the disease (the disease incidence) appears to be greatest for women between 40 and 50 years of age, and for men somewhat later. RA is a chronic disease, and although a spontaneous remission may occur in a very small number of patients, the natural course is almost invariably one of persistent symptoms, waxing and waning in intensity, and a progressive deterioration of joint structures leading to deformations and disability.
The small joints of the cervical spine can also be involved.
Inflammation in the joints manifests itself as a soft, "doughy" swelling, pain, tenderness to palpation and movement, local warmth, and functional impairment. Morning stiffness is often a prominent feature and may last for more than an hour. These signs help distinguish rheumatoid and other inflammatory arthritides from non-inflammatory diseases of the joints such as osteoarthritis (sometimes referred to as the "wear-and-tear" of the joints). In RA, the joints are usually affected in a fairly symmetrical fashion although the initial presentation may be asymmetrical.
Rheumatoid arthritis is a systemic disorder mainly affecting synovial joints. Chemical mediators (Cytokines) released as a result of an abnormal immune reaction triggered by yet undetermined agent/ agents, immune system releases cytokines which gives rise to inflammation of joint synovium (Synovitis). Constitutional symptoms such as fever, malaise, loss of appetite and loss weight are also due to cytokines released in to the blood stream due to an abnormal immune reaction. Vasculitis affecting many other organ systems often gives rise to systemic complications. Most common and disabling clinical feature in Rheumatoid arthritis is chronic, deforming, often symmetrical polyarthritis (affecting multiple joints) due to joint Synovitis triggered by an autoimmune reaction in genetically susceptible individuals.

Note for Collagenases
Collagenases are enzymes that break the peptide bonds in collagen.
They assist in destroying extracellular structures in pathogenesis of bacteria such as Clostridium. They are an exotoxin (a virulence factor) and help to facilitate the spread of gas gangrene. They normally target the connective tissue in muscle cells and other body organs.
Collagenase production can be induced during an immune response, by cytokines which stimulate cells such as fibroblasts and osteoblasts, and cause indirect tissue damage.
SANTYL® Ointment, a Healthpoint, Ltd. product, is the only FDA approved product containing Collagenase as its primary ingredient.

The research appears in the February 26 issue of the Proceedings of the National Academy of Sciences.

Argonne National Laboratory brings the world's brightest scientists and engineers together to find exciting and creative new solutions to pressing national problems in science and technology. The nation's first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state and municipal agencies to help them solve their specific problems, advance America 's scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy's Office of Science.

In figure 1, An atomic-force-microscopy image of collagen fibrils in a collagen fiber.

In figure 2, A view of a rat tail tendon using second-harmonic generation microscopy. The collagen fibers show up in green and red.