POLY(L-ALANYLGLYCINE) - MULTIGRAM-SCALE BIOSYNTHESIS, CRYSTALLIZATION, AND STRUCTURAL-ANALYSIS OF CHAIN-FOLDED LAMELLAE

The biosynthesis of poly(L-alanylglycine) (poly(AG)) was performed in high cell density cultures of recombinant Escherichia coli. The purity of the material was determined by amino acid analysis, elemental anal ysis, and H-1 NMR spectroscopy. Fed batch fermentation increased the y ield of recombinant protein from levels of tens of milligrams per lite r (typical of batch fermentation in rich media) to hundreds of milligr ams per liter. Poly(AG) comprising 64 diads [(AG)(64)] was recrystalli zed from dichloroacetic acid solutions in the form of texture-oriented chain-folded lamellae with a lamellar stack periodicity of 3.2 nm. Th e crystal structure within the lamellar core is similar in general, bu t different in detail, to the antiparallel beta-sheet structure previo usly reported for oriented films of poly(AG) and fibers of Bombyx mori silk fibroin (silk II). The structure consists of polar antiparallel (ap) beta-sheets, with repetitive folding through gamma-turns every ei ghth amino acid (including the fold), stacking with like surfaces toge ther. The wide-angle X-ray diffraction signals index on an orthorhombi c unit cell with a (hydrogen bond direction) = 0.948 nm, b (sheet stac king direction) = 0.922 nm, and c (chain direction) = 0.695 nm. The st acking distance (b-value) is increased by about 3% in comparison with the previously reported structure of poly(AG), owing, we believe, to s teric interaction at the lamellar fold surfaces. Random shears of appr oximately +/-a/4 and shears of +/-c/2 in the ac plane are required to obtain a good fit between the calculated and measured X-ray structure factors.