Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II.
The influence of the bulky and H-bonding Tyr side-chain on its Ala- and Gly-rich environment in Bombyx mori silk fibroin was examined by (13)C cross-polarization magic angle spinning (CP/MAS), static (2)H and (19)F NMR and molecular mechanics calculations. Model peptides of the type (AG)(15) were synthesized with Tyr in a number of different positions, precipitated under conditions favoring either of the two characteristic protein conformations, and the resulting structures were assigned from their (13)C chemical shifts. Dialysis of native fibroin or the simple (AG)(15) peptide from a 9 M LiBr solution against water produces silk I (the structure of silk before spinning), whereas drying from formic acid yields silk II (fibrous structure after spinning). We found that the introduction one or more Tyr into (AG)(15) can have a dramatic effect not only on the local backbone conformation but also on the long-range intermolecular chain packing in the samples. The antiparallel beta-sheet conformation of silk II is able readily to accommodate a single Tyr residue. Interestingly, the beta-turn conformation of silk I only remains stable when Tyr is positioned near the chain terminus in (AG)(12)YG(AG)(2), but the conformation is driven towards silk II when Tyr is located in the central region of (AG)(7)YG(AG)(7). The role of H-bonding was tested by replacing Tyr with Phe or 4F-Phe, which are no longer compatible with silk I and fully induced a silk II conformation. In the presence of several Tyr residues a mixture of distorted beta-sheet and beta-turn conformations was obtained, regardless of the precipitation conditions. Static (2)H NMR of ring-deuterated [3',5'-(2)H(2)]Tyr located in the central region of (AG)(7)YG(AG)(7) showed that the side-chain is immobilized in both silk I and II, which was also observed by static (19)F NMR of the 4F-Phe analogue. To visualize the local packing around the Tyr side-chain, molecular mechanics calculations were performed on a mixture of (AG)(4) and AGAGYGAG, starting from either the beta-turn type II or the antiparallel beta-sheet structure. The resulting structures show that the intermolecular chain arrangement is significantly affected by Tyr, thus explaining the long-range packing effects in the semi-crystalline regions of silk fibers compared with the crystalline regions that are devoid of Tyr.
Asakura
Tetsuo
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184- 8588, Japan. asakura@cc.tuat.ac.jp
Suita
Kohei
K
Kameda
Tsunenori
T
Afonin
Sergii
S
Ulrich
Anne S
AS
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx; D005349:Fibroins / Q000737:chemistry*; D005561:Formates; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D047011:Silk / Q000138:chemical synthesis* / Q000737:chemistry*
D005561:Formates; D010446:Peptide Fragments; D047011:Silk; C030544:formic acid; D005349:Fibroins