The insect proteins, dubbed "projectin" and "kettin," are members of a protein family known as the titins, which includes proteins found in human muscle. Tiny defects in human titins, caused by mutations at single points in their genetic code, have been linked to congenital heart and kidney disorders. Recent studies have shown that the titins are also capable of sensing forces applied to the body and releasing signals that affect genes linked to muscle growth.

The UTMB researchers probed projectin and kettin with a custom-built atomic force microscope. The device uses a gold-coated glass tip about 2,000 times smaller than the point of a pin to pick up single protein molecules. It then stretches and compresses them like miniature springs to directly measure tiny changes in elasticity that correspond to the unfolding and refolding of different parts of the protein.

Information gained from this and similar experiments may greatly advance scientists� understanding of the basic problem of protein folding � the process by which a linear chain of hundreds or thousands of amino acids spontaneously assembles itself into the proper shape to do a specific job in a living organism.

"We�re able to control the shape of single titin molecules for long periods of time basically with a joystick or a mouse, and that gives us a new perspective on the folding and unfolding of a protein with very important physiological implications," said UTMB associate professor of neuroscience and cell biology Andres Oberhauser, senior author of a paper on the work published online today in the Proceedings of the National Academy of Sciences.

Oberhauser and UTMB graduate student Tzintuni Garcia collaborated on the research with scientists from the University of Oxford, the universities of Heidelberg and Muenster, and the European Molecular Biology Laboratory.

"We�re working with insect titins because it simplifies things," Oberhauser explained. "The human titins are very large and complicated, the largest proteins produced by the human genome � but the lessons we�re learning here should translate well to human titin studies."

Projectin and kettin act in insect wing muscles like very stiff springs, a property that lets some insects beat their wings as fast as a thousand times per second. In human muscles and other tissues, titin proteins perform a similar spring-like function, stretching out and springing back to keep heart muscle, for example, constantly expanding and contracting.

Oberhauser�s atomic force microscope measured the tiny, changing forces exerted by these proteins with a minute tip attached to a thin glass rod, whose slightest bending (down to a millionth of a millimeter) could be measured with a laser scanner. Protein samples were then placed on a "positioner" platform whose up-and-down movements could also be controlled to within a millionth of a millimeter. After raising the protein to contact the tip, the researchers slowly pulled the positioner down, measuring variations in the force exerted by the protein on the tip by monitoring how much the rod bent.

"As we stretch the molecule, we can watch its domains � smaller substructures of the protein � unfolding by watching the change in force," Oberhauser said. "We can grab a single molecule and make it unfold, and then turn down the force and watch it refold, then turn up the force and stretch it again, over and over again, hundreds of times. This is really a new avenue to study the protein folding process, and I can see it contributing in all sorts of ways in the future."

Single-molecule studies of titin proteins could yield critical insights into the way genetic mutations alter protein shapes to produce such disorders as hypertrophic cardiomyopathy (excessive thickness of the heart muscle), polycystic kidney disease and tibial muscular dystrophy, as well as titin�s role as a force-sensitive signaling "switchboard" for cells. Beyond titin, Oberhauser said, the technique holds promise as a tool for studying protein misfolding disorders like Alzheimer�s disease and mad cow disease. It could also serve as a source of crucial data for computer models of protein folding, a key tool for rational drug design.

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