Clarifying the basis of collagen's stability The existing explanation for the stability of collagen is incorrect, say researchers at the University of Wisconsin, Madison. Collagen is an insoluble fibrous protein found in connective tissues and in bones. Each polypeptide chain consists of repeats of the sequence XYGly, where Gly is glycine, X is often proline (Pro), and Y is often hydroxyproline (Hyp). The chains are wound in tight triple helices that assemble into strong filaments. Collagen triple helices that contain more hydroxyproline are more thermally stable than others. The increase in stability has been attributed to extra hydrogen bonds between the hydroxyl group of the amino acid and a network of bridging water molecules in the triple helix. This explanation was advanced almost 25 years ago and is supported by X-ray diffraction analysis of crystalline collagen. Former postdoctoral fellow Steven K. Holmgren, graduate student Kimberly M. Taylor, postdoctoral associate Lynn E. Bretscher, and associate professor of chemistry and biochemistry Ronald T. Raines now say the enhanced stability could not be due to extra hydrogen bonding. They reach this conclusion after experiments with a synthetic collagen of 10 ProFlpGly units, (ProFlpGly)10, where Flp is fluoroproline. Fluorine in fluoroproline does not form hydrogen bonds to a significant extent. So if stability were related to hydrogen bonding,fluoroproMe-containingcollagen ought to be less stable. Thermal denaturation studies by Raines and coworkers, however, show that triple helices of (ProFlpGly)10 begin to denature only at 91 °C, compared with 69 °C for (ProHypGly)10 and 41 °C for (ProProGly)10 [Nature, 392,666 (1998)].
Taylor (from left), Raines, that stability of synthetic extra hydrogen bonding.
Trans peptide bonds H N
A
0
O
Proline, X = H Hydroxyproline, X = OH Fluoroproline, X = F
"I was astonished that replacing a hydroxyl group with a fluorine had such a big effect on the stability," says Nick Pace, an expert on protein stability and professor of biochemistry and genetics at Texas A&M University Health Science Center, College Station. And, says Peter A. Kollman, an expert on protein biophysics and professor of pharmaceutical chemistry at the University of California, San Francisco: "This is a very exciting result, showing that collagen stability is not due to water" hydrogen-bonding to hydroxyprolines. The Wisconsin researchers had suspected that hydrogen bonding between hydroxyproline and bridging water molecules was unlikely to contribute to stability because that would mean immobilizing more than 500 water molecules per natural triple helix, an enormous loss of entropy. With their new results, they suggest that the stabilizing effect comes from "previously unappreciated inductive effects." Raines and coworkers believe electron withdrawal by the fluorine in fluoroproline favors the trans orientation required of peptide bonds in triple-helical collagen. Fluorine pulls electrons better than does a hydroxyl group, thus the increase in stability when hydroxyproline residues are replaced with fluoroprolines and when proline residues, which lack an electron-withdrawing group, are replaced with hydroxyprolines. Such an effect "is unlikely to provide more than a few tenths of a kilocalorie per mole per residue," notes Pace. "But since every third residue is a fluoroproline in the [Wisconsin] model, the stability enhancement could be substantial." The finding suggests that collagens with different stabilities can be prepared by modifying the electron-withdrawing effect of the hydroxyl group in hydroxyproline residues. Such modifications are "likely to lead to important new biomateriand Bretscher have shown als," says Raines. collagen is not due to Maureen Rouhi
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