Crystal-clear view of insulin
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Weightless Research has Heavy Implications Crystal-clear view of insulin should lead to improved
therapies for diabetics
July 1998 Super-fine insulin crystals grown in space are giving hope that new forms of the hormone will reduce some of the health risks and discomfort that go with living with diabetes.
The crystals provide scientists with a view of insulin's structure that is equivalent to seeing grains of powdered sugar on a doughnut - instead of just the doughnut - from almost 300 km (180 miles) away. Details have been resolved down to 1.4 Angstrom (1.4 Å)- less than two-millionths the width of a human hair.
Right: The size and optical clarity of space-grown insulin crystals is clearly better than for Earth-grown crystals. But it's under X-rays that they really shine. High-resolution copies of these images are available from the Marshall Image Exchange.
From this, scientists expect to be able to design new forms of insulin that the body can absorb more effectively than the forms now on the market. With certain chemicals bound to insulin, diabetics could inject themselves once every three days or so, instead of one to three times a day.
"The space-grown insulin crystals have provided us new, never-before-seen information," said Dr. G. David Smith, a scientist at Hauptman-Woodward Medical Research Institute, in Buffalo, N.Y. "As a result, we now have a much more detailed picture of insulin."
He conducted this research with Drs. Ewa Ciszak and Walter Pangborn, then both with the Hauptman-Woodward Institute. Ciszak has since joined the Laboratory for Structural Biology at NASA's Marshall Space Flight Center. Their work was described in the August 1996 issue of Protein Science, the journal of the Protein Society. They were supported by grants from NASA and the National Institutes of Health.
Their success also highlights the value of using the weightlessness of space in biotechnology research, a key thrust in NASA's microgravity science.
"From the crystals that came back on the Space Shuttle," said Dr. Larry DeLucas, director of the Center for Macromolecular Crystallography (CMC) at the University of Alabama in Birmingham, "we were able to see details in the three-dimensional structure - the picture of the protein - that we could never have seen with the Earth-grown crystals."
Smith's experiments were conducted through the CMC using the Protein Crystallization Facility that the CMC had developed for use aboard the Shuttle.
DeLucas noted that Dr. Herbert Hauptman, director of the Hauptman-Woodward Institute and a Nobel laureate, "said that it was unlikely that they would ever have been able to understand exactly how the drug was interacting with the protein, insulin, without those space grown crystals."
Right: Wireframe models, produced by computer, depict the structure of insulin molecules. The space-grown crystals let scientists fill gaps in their understanding of the structure, and will lead to improved drugs for diabetes therapies. High-resolution copies (1,000 pixels wide) of the Earth-grown model and space-grown model are available from NASA/Marshall.
Insulin is a lifelong treatment - not a cure - for diabetes, a breakdown in an important control system in the human body. The human body has many "feedback" controls like this to regulate the chemicals that make our bodies work.
For sugar, the basic fuel of life, the body produces insulin, a hormone that regulates the metabolism of sugar. Insulin circulates in the bloodstream and interacts with all cells in the body. It's a little like using the accelerator to maintain a car's speed. You press the accelerator a little if the speedometer drops off, and ease off on the pedal if the speedometer goes too high.
Like any control system, it can be knocked out of kilter or shut down altogether. This has happened to an estimated 5 to 10 million Americans with Type 1 diabetes. A bizarre failure in the immune system directs the body to kill its own Beta cells (in the pancreas, behind the stomach), the ones that sense sugar levels and make insulin. Suddenly, you can't see the speedometer or touch the accelerator. Sugar cannot reach cells, and the victim drinks large quantities of water to pass the excess sugar: diabetes means "to pass through." (Type 2 diabetes occurs, often in older and obese people, when the body develops a resistance to insulin.)
For centuries, diabetes was a death sentence and the victim starved to death in the midst of plenty - until the 1920s.
In 1920, Drs. Frederick Banting and C.H. Best of Toronto, Canada, discovered insulin. In 1922, they demonstrated that insulin refined from animals and injected into humans could control the disease. Although many advances have been made since then - including genetic engineering to produce human insulin - the treatment remains basically the same. The diabetic must check his or her blood sugar level, and inject enough insulin to match their activity, planned meals, and metabolism.
Shots are not a "one dose fits all" treatment. They carry some risk because they are no match for the exquisite feedback control that a healthy pancreas provides. Shots are more like goosing the accelerator a few times a day, letting the car coast, and hoping that the speed averages out just right.
It rarely does, meaning that diabetics face deteriorating health as the surplus sugar damages fine arteries and veins, leading to kidney failure, blindness, and loss of fingers and toes and even legs.
All told, Types 1 and 2 diabetes and its effects account for about $1 of every $7 spent on health care in the United States, and for a significant number of premature deaths.
Left: Missions aboard the Space Shuttle - using both Spacelab (shown), Spacehab, and other facilities - have provided lessons on how best to grow protein crystals in microgravity, and produced crystals yielding key insights into a number of protein structures.
Because of its importance to human health, insulin has been one of the most studied biological molecules in modern medical history. While the major structures and components have been worked out over the years, important details have eluded scientists seeking a more effective way of delivering the hormone once it is injected into the body.
"What we're looking for, long-term, is a time release formulation of insulin," said Dr. Marianna Long, associate director of the CMC in Birmingham. "And what this means for the patients is fewer shots per day, and maybe fewer shots per week, and their lifestyle would be a lot easier."
Insulin comes in two basic forms called monomer and hexamer. The first is a lone insulin molecule that is absorbed rapidly by the body and is not very stable when stored. Diabetics inject themselves with the hexamer version - six molecules linked in a tight association - that gradually dissolves to deliver the monomers into the bloodstream.
Even within the hexamer form, insulin has three different configurations, and that offers the potential for developing a new form of insulin with an even slower delivery rate.
"Insulin has this phenomenal property called allosterism," Smith explained. "It can change its shape spontaneously yet still be the same molecule." It's like folding and extending your arms and legs: you take different shapes, but are still the same person.
The normal insulin hexamer is called T6, meaning that all six molecules have a particular shape. Under certain conditions, molecules will change their shape to form a hexamer called T3R3. R6 forms are also possible and their structures have been studied, but so far, no other forms are known to exist.
"With a T3R3 insulin," Smith explained, "a diabetic could have a longer basal level of insulin." A single dose would provide a stable level for three or four days and smooth the highs and lows that normally come with daily shots.
Smith said that a number of chemicals can cause the T6-to-T3R3 flip, "But we don't know why insulin wants to do this."
Smith said that insulin has a number of subtle design points that still are not fully understood, so the significance - or insignificance - of changing certain parts remains unknown.
"It's an amazing molecule," Smith said. "In certain places the molecule is very forgiving to changes. But if you make too big a change, you wind up with a molecule that is inactive."
Shape, size, and position matter in large biomolecules. These must be known in order to understand how the molecules work. And that has been a challenge that the space program has helped scientists to meet.
Like many chemicals in the body, the three dimensional structure of insulin is not just complex, but flexible, like a flowery bundle of ribbons on a birthday present. The ribbons may be stapled in place, but they can flex and shift, taking slightly different shapes.
And that has frustrated scientists using one of their most powerful tools, X-ray crystallography. X-rays passing through a crystal produce diffraction patterns that correspond to the locations of atoms, and even the electron clouds surrounding the atoms, making up a molecule. With enough patterns made from different angles, scientists can decode the structure of the crystal - if they start with a good specimen.
Large biomolecules can flex under their own weight and as more molecules link up, leading to a crystal that has a lot of internal disorder. And that produces an image of the crystal structure that is, in effect, out of focus.
This is where the space program has made a vital contribution to national health.
"Going into space will give us crystals that are vastly superior to those that we grow here on Earth," Long said. "They're bigger, they have better optical clarity, they look better. But they can give us the information that we need, wonderfully detailed information that we can never get from crystals grown here on Earth. And with this information we can develop better medicines."
In the microgravity environment of space, most materials will form crystals with more uniform structures than they will on Earth. The crystals are not always larger, but size is not everything. In X-ray crystallography, what counts is internal order, like having platoons of soldiers lined up for parade.
For this insulin study, these well-ordered crystals were produced by Protein Crystallization Facility during eight days in space aboard the Shuttle Discovery on the STS-60 mission in 1994.
After the return to Earth, the new crystals are subjected to studied by X-ray crystallographic techniques at the Hauptman-Woodward institute.
The result was an exceptionally detailed map of the insulin molecule in the T3R3 hexamer form. Previous images, using Earth-grown crystals, resolved features down to about 1.9 Å showed structures that seemed to hang in space by themselves. Without knowing how they connected, scientists could only guess at their purpose.
In the new, 1.4 Å image, the scientists could see better how structures attach. From this, they will to see more details and be able to design chemicals which will spontaneously generate T3R3 hexamers that are stable when stored, yet dissolve at just the right rate in a diabetic's body.
At least three years of research and trials must follow before the new knowledge translates into improved therapies, but DeLucas is hopeful.
Right: A mockup of the Microgravity Science Glovebox that will see a number of biotechnology experiments, include Protein Crystal Growth, aboard the International Space Station.
"We don't know what the long-term effects will be, yet," he said, "the basic research will bear that out along with the clinical studies - but it may be that having a more steady release of the insulin may ultimately decrease the complications due to diabetes."
And DeLucas is optimistic that the results from the Shuttle-based insulin experiments bode well for long-term protein crystal growth experiments aboard the International Space Station which will include a biotechnology facility among its many experiments.
"The key advantage of the International Space Station is the fact that we will be able to do science all year long," DeLucas said. "We won't have just a glimpse at it, as we do with the Space Shuttle. You put your experiment up there and for a long period of time we'll be able to do successive experiments, just as we do here on Earth. That's the only way to make rapid progress in any scientific field."
The Hauptman-Woodward Medical Research Institute in Buffalo conducts research on a number of medical fronts in addition to diabetes.
NASA/Marshall's Macromolecular Crystal Studies are detailed by Dr. Eddie Snell.
The Center for Macromolecular Crystallography at the University of Alabama in Birmingham describes its work in space-based protein crystal growth.
On target for a cure discusses protein crystal growth experiments aboard the Microgravity Science Laboratory-1 mission in 1997.
The American Diabetes Association has details bout the disease, its treatment, and how to live with it.
The Banting Museum and Education Centre in Canada describes Dr. Frederick Banting's discovery on insulin and research on diabetes.
The August 1996 issue of Protein Science, published by The Protein Society, carried Smith's article on high-resolution studies of insulin grown on STS-60: "A Novel complex of phenol derivative with insulin: Structural Features related to the T -> R transition." Protein Science, (1996), 5:1502-1511.
Working with NASA - Technology Transfer Office
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