They hold your skin together; they make your muscles move; they fight infections; and they allow you to see. Yet proteins rarely inspire a thought from the average person. Yet when things go wrong with proteins in the body, bad things happen – like cancer, diabetes and a host of other medical problems.
Fortunately, a team of University of Arkansas researchers uses cutting-edge techniques and equipment to study these molecules at the atomic level, and their work ties together medical advances with a fundamental understanding of these important structures. By getting closer to the knowledge of how proteins work, they are better able to design medical interventions for when they don’t work.
“Proteins do all the functions that are needed in a biological system,” said Frank Millett, University Professor of chemistry and biochemistry and the director of the Center for Protein Structure and Function. He checks off a brief list of things proteins do in the human body — act as signals between the brain and muscles, carry oxygen in the blood, allow the brain to function. “When something goes wrong, it’s often a protein function that has gone wrong.”
The National Institutes of Health has invested about $20 million in the center, starting in 2000 and continuing through 2010. Using this funding, the center provides funding to junior researchers, who work on their research projects with senior researchers as advisers. In all, 22 scientists conduct research through the center. Their research focuses on five major projects, all of which examine a key aspect of protein structure and function. While the projects show great diversity in their nature, they all share a common theme — better understanding of the basic processes of living things in order to enhance the quality of life for everyone.
A Binding Finding
The protein collagen holds many things together in the human body, including the tissue in our skin. Scientists estimate that 25 percent of the total protein content in a body is composed of collagen. Collagen is found in ligaments, bones, tendons and other connective tissues. When it deteriorates, wrinkles appear in the skin, which is why collagen injections to reverse the process have become popular.
While many people know about the protein because of its cosmetic properties, chemistry professor Joshua Sakon looks at collagen in a different way. He studies the destruction of collagen by a protein called collagenase, which binds to collagen in the presence of calcium and destroys connective tissue.
This collagenase, harbored by a particular type of bacteria, contains at least one collagen-binding domain, which allows it to bind to the collagen before destroying it. Sakon and his colleagues are closely examining this binding domain to see how it might be used to deliver drugs to connective tissue.
Sakon uses X-ray crystallography to examine the structure of collagen and the collagen-binding domain. The researchers create a crystal of the protein they wish to examine, then place it in an X-ray beam. The X-rays produce a diffraction pattern that is related to the structure of the atoms in the crystal. The crystal is rotated in the X-ray beam to get patterns in three dimensions. The computer collects the data, which is merged with other information about the protein, including its known sequence of amino acids. All the information combined allows the researchers to look at a three-dimensional crystalline structure of the protein in question.
In the case of the collagen-binding domain, the researchers wanted to determine its structure to see if they could create an efficient delivery system for different kinds of hormones needed in collagen-rich tissues.
“By adding the collagen-binding domain, the hormones actually stay at the site” where they are needed, Sakon said.
After determining its structure, Sakon and his colleagues attached a bone growing hormone to the collagen-binding domain. They worked with Robert C. Gensureto of Ochsner Hospital in New Orleans and Osamu Matsushita of Kitasato University Medical School in Japan to inject the modified hormone into mice. The mice showed a 15 percent improvement in bone mineral density in a month. This technique potentially could be used to treat osteoporosis, a disease characterized by decreasing bone density over time.
“We have made an effective vehicle for the bone-growing hormone,” Sakon said. The university has applied for a provisional patent for this research. Sakon and his colleagues continue to look at other ways to use the collagen binding domain as a drug delivery vehicle.
The First Line of Defense
Most people have heard of the adaptive immune system and how it uses antibodies to generate a defense against disease. But Denise Greathouse, assistant professor of chemistry and biochemistry, studies the components of the more ancient innate defense system — which depends on peptides to generate a rapid and immediate defense against foreign invaders. These small proteins are present in tears and other bodily secretions. They help prevent infections when your child scrapes her knee on the sidewalk or when you slice a finger with a kitchen knife.
“They represent your first line of defense against infection,” Greathouse said.
Greathouse studies the properties of lactoferricin, a peptide found in different kinds of mammals’ milk. The peptide has been shown to deter a wide range of disease-causing microbes, and Greathouse and her colleagues seek to enhance those properties to create a possible replacement for some of today’s antibiotics. To do so, they need to overcome a major hurdle:
“It’s not really understood how these antimicrobials work,” she said.
Additionally, even though they are smaller than proteins, peptides can be expensive to make. The peptide lactoferricin only has 25 amino acids, the fundamental building blocks of proteins, but creating an abundant source of this peptide would be costly. A sequence of only six amino acids from lactoferricin has been identified that retains the antimicrobial properties, making it less expensive to manufacture and therefore more amenable to potential drug development.
An important question is: Why does this peptide work specifically against bacterial cells and not human cells? It turns out that the peptide, which has a net positive charge, is selectively attracted to negative charges on bacterial cell membranes. Human cell membranes have no net charge, so there is no attraction between the peptide and the cells, leaving them unharmed.
The peptide is attracted preferentially to the bacterial cell membrane, but the actual mechanism by which the cell is destroyed is not well understood. To find the answer, Greathouse and her colleagues use solid state nuclear magnetic resonance spectroscopy to look at the interactions between the peptide and a lipid bilayer, which consists of fatty acids that mimic a bacterial cell membrane.
Initially they labeled the peptide so they could see how strongly it interacted with the lipids. Their results showed that the interaction of the short peptide was fairly weak. To enhance the interaction of lactoferricin with the lipids, the researchers decided to add fatty acid tails composed of 2-, 4-, 6- or 12-carbon atoms to the peptide. The idea was that the fatty acids would act as “anchors,” holding the peptide at the bacterial membrane. All the fatty acid modified peptides increased lipid binding, but the 12-carbon peptide interacted most strongly. It completely destroyed the lipid membrane.
Enhancing the antimicrobial properties of this sequence is one of the primary goals of Greathouse’s research.
“By adding fatty acid chains, we were able to increase both the membrane interaction and the antimicrobial activity of the peptides,” Greathouse said. The next question is: How do the newly synthesized peptides affect human cells? The researchers will next test the modified peptides with mammalian blood cells to see how they are affected. This test is crucial, because an effective antibiotic must selectively kill bacterial cells while leaving human cells unharmed.
Ready, Set, Grow!
In the beginning, we are all just a blob of cells. What tells these cells to form a liver, create a finger or organize into brain tissue are proteins called fibroblast growth factors. In addition to directing the growth of various body parts and internal organs, these factors heal skin torn by wounds or tissue sliced by surgery.
Suresh Kumar, an assistant professor of chemistry and biochemistry, studies these factors to better understand how they function — and what can go wrong.
“Fibroblast growth factors can be a boon or a bane,” Kumar said. “A normal cell knows when to stop growing, but a cancer cell does not know when to stop growing.” One way to block cancer growth may be to inhibit fibroblast growth factors.
The millions of proteins found in a cell all have receptors for their destined functions, and fibroblast growth factors are no exception — they must bind with their receptors in order to function. Chemical signals allow the factors to find their receptors amid the chaos.
“By understanding the signals between the receptor and the fibroblast growth factors, we would be able to intervene with that particular signal,” Kumar said.
Kumar uses nuclear magnetic resonance spectroscopy to study the three-dimensional structures of the fibroblast growth factor protein in solution. Through his studies of these structures, he has developed molecules that may be the first generation of drugs that can block fibroblast growth factor receptors, possibly shutting down the uncontrolled cell growth that characterizes cancer.
“Sometimes we want to block FGF. But sometimes we want to support its stability,” Kumar said. In the case of healing wounds, for instance, it would be helpful to have fibroblast growth factors work fast and efficiently. To increase the efficiency of fibroblast growth factors, researchers have to understand the route that it takes to reach receptors located on the outer surface of the cell membrane. Studies in Kumar’s group has showed that fibroblast growth factor follows unconventional routes to reach its receptor. The growth factor does not have a signal peptide, a set of amino acids that tells it where to go, yet it has to reach its receptor, Kumar said.
“It’s like forgetting to write the address on an envelope, but the letter gets there anyway,” he said.
It appears that the fibroblast growth factor forms a multi-protein complex that allows it to travel to its final destination, where it will promote cell growth. Kumar’s group is now studying this complex to better understand its role in the vital healing process. This unusual pathway may turn out to be a model to understand the unconventional secretion of other proteins that lack signal peptides.
Chemistry and biochemistry professor Matt McIntosh wants to mimic nature in his laboratory. Specifically, he studies ways to synthesize or mimic chemical compounds found in nature that have biomedical properties.
“Many desirable compounds found in nature are only available in small quantities,” McIntosh said. He and his colleagues are working on synthesizing a compound called sclerophytin that scientists believed to have anti-cancer properties. Small amounts of sclerophytin have been found in certain types of soft coral in the ocean, but not in quantities large enough to be used as a drug. In addition to trying to synthesize the compound itself, the researchers have created analogs, molecules with structural similarities to sclerophytin. These analogs are typically easier to make, requiring only three or four chemical reactions instead of 20 or more steps.
The researchers have created about 20 analogs for sclerophytin. Working with researchers at the University of Arkansas for Medical Sciences, they found that some of the analogs inhibit cancer cell growth.
Meanwhile, McIntosh still seeks to synthesize sclerophytin, a process that will take 20-25 steps. However, the plot has thickened: The most current research on the molecule suggests that it may not have strong anti-cancer properties as was previously thought.
“That makes it all the more interesting that ‘sclerologs’ do appear to have these properties,” he said.
Two other groups have synthesized sclerophytin, but not in the same way, and that is crucial.
“Each group will discover new chemistry that is a consequence of the way they assembled the molecule,” McIntosh said.
The researchers have obtained a provisional patent for the analogs to sclerophytin and plan to continue studies on the anti-cancer properties of these compounds.
Plants gather light and convert it into energy. To do so, light-gathering proteins must find their way into the chloroplasts, the green, membrane-surrounded compartments that serve as plant powerhouses. But how do the light-gathering proteins know where they are supposed to go?
Biological sciences professor Robyn Goforth studies the answers to this question by looking at the signal recognition particle pathway, which is responsible for taking proteins from where they are made to where they are used. This pathway occurs in mammals, bacteria and plants, with minor but significant differences between the three. Of the three, the pathway in plants has been the least studied.
Goforth and her colleagues have examined a receptor in plants and bacteria that behaves differently than it does in mammals; in mammals, the receptor remains attached to the membrane at all times, but in plants and bacteria, the receptor seems to alternate between attachment to and roaming off of the membrane.
The receptor protein in question has three parts, two of which are fairly well mapped. The third part seems to change depending upon the organism. So Goforth and her colleagues decided to study what would happen if they made changes to the protein.
“We started by chopping off pieces,” she said. The pieces in question were amino acids, which form the building blocks of a protein. They wanted to see if changes in the unknown region of the receptor protein would affect its ability to move a specific protein from point A — the solution — to point B — the cell membrane.
To do this, they isolated chloroplast membranes from pea plants, then introduced the modified proteins, first taking off three amino acids, then six, then nine. They then examined the modified proteins’ ability to move light-harvesting proteins to the membrane.
“We found that we could chop off three amino acids or six amino acids with no problem. But after we chopped off the next six, the activity went down to almost nothing,” Goforth said.
Further studies looked at the location of the receptor protein. They found that the unmodified protein spent half of its time on the membrane and half in solution. However, the modified protein that lost its function couldn’t get to the membrane, and therefore it couldn’t do its job.
“We can trace this loss of activity to a single amino acid,” Goforth said. That amino acid, phenylalanine, is paired with another phenylalanine, and this pairing is also found in receptors from other plant and bacterial species.
Goforth teamed up with biological sciences professor Ralph Henry and chemistry and biochemistry professor Suresh Kumar to examine the structure of the protein when it interacts in the membrane and in solution. They found that this region of the receptor protein had different structures in the two different environments.
“When you change the phenylalanine, you don’t get the structural switch,” Goforth said. “This peptide is both necessary and sufficient for targeting proteins to the membrane.”
Fighting diseases like osteoporosis, creating better antibiotics, discovering new ways to combat cancer – University of Arkansas researchers in the Center for Protein Structure and Function continue their basic research in hopes of contributing to the body of biomedical knowledge in a fundamental way. The findings they make today may become the basis for the pharmaceuticals you’ll be putting in your medicine cabinet 10 years from now.