Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence

Citation
Yt. Zhou et al., Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence, BIOMACROMOL, 2(1), 2001, pp. 111-125
Citations number
70
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics","Organic Chemistry/Polymer Science
Journal title
BIOMACROMOLECULES
ISSN journal
1525-7797 → ACNP
Volume
2
Issue
1
Year of publication
2001
Pages
111 - 125
Database
ISI
SICI code
1525-7797(200121)2:1<111:GDSASA>2.0.ZU;2-J
Abstract
The flagelliform silk protein underlies the unique elastomeric properties d isplayed by the capture spiral of arachnid webs. To investigate molecular m echanism underlying the elastomeric recovery of the capture spiral, a model polypeptide based upon the elastomeric repeat sequence of Nephila clavipes flagelliform silk protein has been synthesized using recombinant DNA techn iques. Polypeptide 1 contains 11 repeats of the 25 amino acid sequence [(Gl y-Pro-Gly-Gly-Ser-Gly-Pro-Gly-Gly-Tyr)(2)-Gly-Pro-Gly-Gly-Lys] and was expr essed in Escherichia coil strain BL21(DE3) as a C-terminal fusion to a deca histidine leader sequence. A combination of H-1-H-1 COSY, DEPT,H-1-C-13 HET COR, and H-1-C-13 HMBC NMR spectroscopy was employed on polypeptides 1 and the [1-C-13]glycine-labeled analogue 1G to assign the H-1 and C-13 NMR reso nances of the amino acid residues comprising the flagelliform silk repeat s equence. The conformational properties of 1 in aqueous solution were invest igated using a combination of CD, FTIR, VT-NMR, and two-dimensional NOESY N MR. These techniques were consistent with the presence of small but detecta ble population of beta -turn conformers between Gly(1) and Gly(4) of the pe ntapeptide units of 1. FTIR and CD studies of solid films of 1 indicated an increase in beta -turn population in the solid state, which coincided with the decrease in hydration level of the polypeptide. The spectroscopic info rmation suggests that the pentapeptide segments of the flagelliform silk pr otein adopt a beta -turn conformation in the fiber and that the mechanism o f elasticity may resemble that proposed for other beta -turn forming polype ptides including elastin.