The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important.
Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα)8TIM barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS.
The generality of these observations for βα-hairpin clamps in TIM barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 Å make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM barrel proteins was examined by a survey of 71 TIM barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-barrel.
Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the barrel have evolved to render a subset of the clamp interactions redundant. Distinct sequence preferences for the partners in the βα-hairpin clamps and the neighboring segments may be useful in enhancing algorithms for structure prediction and for engineering stability in TIM barrel proteins.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1433 |
| Date | 01 July 2009 |
| Creators | Yang, Xiaoyan |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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