Synthesis and Characterization of Periodic Polypeptides Containing Repeating —(AlaGly)_xGluGly— Sequences

We have expressed in E. coli a series of periodic polypeptides represented by sequence 1. Our objective has been an understanding of the role of chemical sequence in determining the chain folding behavior of periodic macromolecules. Molecular organization has been examined by infrared spectroscopy and ^1H and ^(13)C NMR methods and a preliminary model of the folded structure has been developed

Periodic Polypeptides Based on Poly(L-Alanylglycine): Biological Synthesis and Verification of the Structure of A Series of Polymers Containing Tandem —(Alagly)xGlugly— Repeats

The experiments reported herein constitute part of an investigation of the relationship between primary sequence and higher order structure in repetitive polypeptides. Three series of artificial DNAs encoding poly-peptides represented by the general formula + (GlyAla)_nGlyGlu]_m— were constructed and cloned in Escherichia coli.Protein expression was monitored by in-vivo labeling with ^3H-glycine. Protein products were isolated from the cell lysates by stepwise acidification in yields of 20–50 mg/L of culture, and fusion fragments derived from the cloning vectors were removed by cyanogen bromide cleavage. Amino acid sequence analysis verified the expected sequences through the first 40–50 N-terminal residues

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed

Crystal Structures of Chain-Folded Antiparallel β-Sheet Assemblies from Sequence-Designed Periodic Polypeptides

The crystal structures and textures of a family of sequence-designed periodic polypeptides were investigated and analyzed using X-ray diffraction, vibrational spectroscopy, and cross-polarization magic angle spinning ^(13)C nuclear magnetic resonance. The repetitive amino acid sequences are described by −[(AG)_xEG]−, with integer x from 3 to 6. These macromolecules were prepared via bacterial expression of artificial genes and are monodisperse. Crystalline samples were obtained, and the interpretation of the X-ray diffraction results was aided by the generation of computer-simulated X-ray diffraction patterns. This allowed direct comparisons to be made with the observed texture-oriented X-ray diffraction photographs. All diffraction and spectroscopic evidence supports an antiparallel (ap) β-sheet structure, and all structures index on orthorhombic sublattices similar to those reported for Bombyx mori silk fibroin and poly(L-alanylglycine). The unit cell parameters for poly(AG)_3EG, for example, are a = 0.948 nm (hydrogen-bond direction), b = 1.060 nm (ap β-sheet stacking direction), and c = 0.695 nm (chain direction). Selective line broadening is observed for wide-angle diffraction signals with l ≠ 0 (for the 211 in particular) and gives an estimated crystal size of <4 nm in the chain direction. This, coupled with the appearance of a low-angle particle interference peak at 3.6 nm, indicates a crystal size over an order of magnitude less than the chain length and suggests an adjacent reentry chain-folded lamellar structure incorporating the ap β-sheet architecture. A structure with polar ap β-sheets and γ-turns, stacking with the hydrophobic methyl groups of the alanyl residues in contact, is selected by X-ray structure refinement to give the best match with the experimental data. The pattern of crystallization behavior of the poly(AG)_xEG family is consistent with the folding periodicity being in-phase with the amino acid sequence so that the glutamic acid residues are confined to the lamellar surfaces

In Vivo Synthesis and Structural Analysis of Alanylglycine-Rich Artificial Proteins

A series of 22 alanylglycine-rich artificial proteins has been prepared by bacterial expression of the corresponding synthetic genes. These polymers have been isolated and purified, and subjected to structural analysis by gel electrophoresis, mass spectrometry, x-ray scattering, and vibrational and NMR spectroscopy. Strategies have been developed for the in vivo synthesis of artificial proteins containing non-natural amino acids