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J. Biol. Chem., Vol. 282, Issue 41, 29757-29765, October 12, 2007

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Mechanism of Stabilization of a Bacterial Collagen Triple Helix in the Absence of Hydroxyproline^*

Angela Mohs, Teresita Silva, Takeshi Yoshida, Ravish Amin, Slawomir Lukomski, Masayori Inouye, and Barbara Brodsky¹

From the Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 and the Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia 26506

The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)₇₉, which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)_n sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 °C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 °C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.

Received for publication, May 15, 2007 , and in revised form, August 9, 2007.

^* This work was supported by National Institutes of Health Grants R21EB007198 and GM60048 (to B. B.) and Grant AI50666 (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-4397; Fax: 732-235-4783; E-mail: brodsky{at}umdnj.edu.

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