title: Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence.
abstract: The flagelliform silk protein underlies the unique elastomeric properties displayed by the capture spiral of arachnid webs. To investigate molecular mechanism 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 techniques. Polypeptide 1 contains 11 repeats of the 25 amino acid sequence [(Gly-Pro-Gly-Gly-Ser-Gly-Pro-Gly-Gly-Tyr)(2)-Gly-Pro-Gly-Gly-Lys] and was expressed in Escherichia coli strain BL21(DE3) as a C-terminal fusion to a decahistidine leader sequence. A combination of (1)H-(1)H COSY, DEPT, (1)H-(13)C HETCOR, and (1)H-(13)C HMBC NMR spectroscopy was employed on polypeptides 1 and the [1-(13)C]glycine-labeled analogue 1G to assign the (1)H and (13)C NMR resonances of the amino acid residues comprising the flagelliform silk repeat sequence. The conformational properties of 1 in aqueous solution were investigated using a combination of CD, FTIR, VT-NMR, and two-dimensional NOESY NMR. These techniques were consistent with the presence of small but detectable population of beta-turn conformers between Gly(1) and Gly(4) of the pentapeptide 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 information suggests that the pentapeptide segments of the flagelliform silk protein adopt a beta-turn conformation in the fiber and that the mechanism of elasticity may resemble that proposed for other beta-turn forming polypeptides including elastin.
authors.affiliation: Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: S
authors.initials: S
authors.lastname: Wu
authors.forename: V P
authors.initials: VP
authors.lastname: Conticello
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000096:biosynthesis* / Q000737:chemistry*; D002942:Circular Dichroism; D004586:Electrophoresis; D004926:Escherichia coli; D005285:Fermentation; D015870:Gene Expression; D019476:Insect Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D047011:Silk; D013054:Spectrophotometry, Atomic; D017550:Spectroscopy, Fourier Transform Infrared; D013112:Spiders
chemical_list: D000577:Amides; D001704:Biopolymers; D019476:Insect Proteins; D011522:Protons; D011994:Recombinant Proteins; D047011:Silk