Pulling strings: stretching proteins can reveal how they fold.

Proteins, long strings of amino acids, spontaneously fold into intricate shapes that enable them to perform a cell's dazzling variety of functions. To better understand the forces that determine these shapes, scientists have developed a technique for stretching a protein to follow in reverse the path it took when folding.

"The basic idea is to pull the molecule at both ends to stretch it and see what happens," says Ching-Hwa Kiang, a biological physicist at Rice University in Houston.

When a cell builds a protein, it links amino acids that pivot around each other and interlock. These movements are dictated by electrostatic forces between the amino acids and by their tendency to hide their water-repelling sides while leaving their water-loving sides exposed.

A fully folded protein is in a state of minimum energy because force must be applied to pull it apart. Kiang and her Rice collaborators devised a technique to measure that force. They placed water droplets containing proteins on a movable surface below a microcantilever akin to a tiny diving board. The researchers fished for proteins by varying the distance between the surface and the cantilever. When the cantilever snagged one end of a protein, the scientists could pull back the surface, slowly unfolding the protein.

The bending of the cantilever indicated the force required to stretch the protein. The researchers tested their technique on a synthetic version of the muscle protein titin, consisting of a chain of eight identical amino acid strings. As the researchers stretch ed the protein, the strings unfolded one after the other, generating the same sequence of force measurements each time. The team reports its findings in an upcoming issue of Physical Review Letters.

Unfolding a protein requires energy to overcome friction between molecules in addition to the energy needed to counter molecular forces. To tease apart these effects, the team used a mathematical technique invented in 1997 by Christopher Jarzynski, now at the University of Maryland at College Park. That analysis took into account the reductions in measured force due to random molecular jiggling that sometimes kicked the protein into an unfolded state.

The researchers plan to apply their technique to other proteins. They also hope to measure the energy required to unzip the double helix of DNA. Kiang says that researchers could also use the technique to test whether environmental conditions such as acidity or temperature affect folding. Scientists believe that misfolded proteins may cause certain diseases, including Alzheimer's.

Kevin Plaxco of the University of California, Santa Barbara says that scientists are eager to find methods for mapping the energy of proteins. While the new technique traces only one possible way that a protein unfolds, as opposed to the full range of a protein's possible states, "it's the most concrete example I've seen," of such a measurement, he says.