Lots of movement in a small space
Actually, the two requirements should be mutually exclusive: it takes a great deal of effort to bring proteins into the highly ordered crystalline state in order to be able to study their structure, but movement is often essential for the normal activity of enzymes. Ironically, an enzyme that multiplies the DNA in living cells manages the feat of continuing to work in the crystal assembly. Biochemists at the Medical Center at Duke University wanted to investigate the structure of the enzyme DNA polymerase - a protein that catalyzes the replication of genetic material - with the best possible spatial resolution. To this end, they prepared crystals of the active enzyme and subjected them to an X-ray structure analysis (Nature, 15. January).
Most protein molecules are so small that they cannot be seen with the naked eye. At the same time, they are so large that the position of their many atoms cannot simply be calculated theoretically, but must be determined experimentally. Of the various methods commonly used, X-ray structural analysis provides the most accurate results. In this process, crystals of the protein are irradiated with intense X-ray light. From the image created behind the crystal, scientists can then calculate the structure with atomic resolution.
Lorena Beese and her colleagues used this technique to determine the structure of DNA polymerase earlier. In their new work, they wanted to show how the enzyme binds the individual building blocks of the genetic material and, so to speak, "holds it in its hands".
Although researchers around the world have been studying DNA polymerase for decades, not all questions have been answered. The enzyme is part of a complex molecular machinery that helps the cell duplicate its DNA. To do this, the two strands of the double helix are first separated from each other. The DNA polymerase binds to a single strand and, based on its template, assembles the appropriate building blocks, the nucleotides, to form a new second strand. After attaching each individual nucleotide, the enzyme slides a little further on the DNA, thus making relatively large movements. In addition to this copying work, DNA polymerase also proofreads its own work and stops in the event of an error so that repair enzymes can intervene. This system ensures the extremely low error rate that must be maintained for living organisms and their propagation.
Beese's team wanted to capture the intermediate catalytic steps on a DNA polymerase from the bacterium Bacillus stearothermophilus in individual snapshots. So they made crystals of the enzyme that were spiked with DNA and the necessary nucleotides."We were thrilled when we got the first crystals in which the enzyme had bound the DNA," said Beese. "But we were also amazed because only part of the structural data corresponded to what we had expected to see." This is because the DNA polymerase had no nucleotide in its active center. So the scientists had not succeeded in creating the state just before the actual catalytic step.
"Initially we were very disappointed after decoding the structure," said Beese. "But what we found was that the nucleotide was actually incorporated into the crystal. It was fascinating because we realized that the enzyme had retained its catalytic activity in the crystal.” In fact, by the time the measurement was taken, the polymerase had already processed the nucleotide and pulled the DNA a little further. Although activity in the crystals of other enzymes has already been detected, they were not associated with such large movements."With this enzyme, not only does the chemistry work correctly, the DNA template and the new DNA also have to be shifted quite a bit. And that was really unpredictable.”
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