Details

# Nanoscale scaffolding by folding of monodisperse and sequentially precise poly((alanine-glycine)(3)glutamic acid-glycine(glycine-alanine)(3)glutamic acid-glycine): Biosynthesis and characterization by X-ray diffraction, FTIR and NMR

Abstract (Summary)
We have designed repetitive polypeptides which would self assemble into a unique three dimensional structure. The de novo design of these polypeptides is based on existing information on protein chain folding. As a first step to designing more complicated scaffolds, we are interested in designing repetitive polypeptides which would self assemble into lamellae of uniform, predetermined thickness. The design of the polypeptide repeating unit has been based in part on work on the structure of silk (and its analogues) and in part on the literature on reverse turns in globular proteins. The polypeptides in this work comprise alternating beta sheet forming segments and turn forming segments with exact periodicity. Repetitive alanyl-glycyl diads are known to form a beta sheet structure. It is anticipated that these would reside in the lamella and define its thickness. The stronger the thermodynamic driving force for the chains to form alternating beta sheets and turns, the more is the probability of obtaining a unique scaffold. In this thesis, DNA sequences encoding the synthesis of repetitive units of A, B, C and D have been synthesized. Polypeptides with ten repeats of A, four repeats of D and three repeats of C were successfully synthesized in E. coli.\eqalign{{-}(\rm AlaGly)\sb3GluGly(GlyAla)\sb3GluGly{-}&\qquad{\bf A}\cr{-}(\rm AlaGly)\sb3AspGly(GlyAla)\sb3AspGly{-}&\qquad{\bf B}\cr{-}(\rm AlaGly)\sb3GluGly(AlaGly)\sb3ValGly{-}&\qquad{\bf C}\cr{-}(\rm AlaGly)\sb3GluGly(AlaGly)\sb3MetGly{-}&\qquad{\bf D}\cr} The polypeptide containing ten repeats of A (referred to as A-10 in the Abstract) has been characterized in the solid state by x-ray diffraction, FTIR and NMR. The polypeptide A-10 forms an antiparallel beta sheet structure when stirred in 70% formic acid; electron microscopy shows the morphology of the crystallites to be needle like. The unit cell deduced from the diffraction patterns is similar to those from previous diffraction patterns on silks and silk-like polypeptides. The unit cell which best explains the diffraction data for A-10 is monoclinic with a = 9.56 A, b = 10.68 A and c = 7.0 A with the angle between b and c equal to 80$\sp\circ$. FTIR spectroscopy on crystalline A-10 shows the polypeptide predominantly adopting the antiparallel beta sheet structure. The repetitive polypeptide A-10 and a $\sp{13}$C=O enriched analogue have been synthesized biologically in Escherichia coli. The two analogues have been blended in solution and co-crystallized. FTIR spectra (in the amide I region) have been studied as a function of blend composition. Solid state CPMAS NMR experiments were carried out on the amorphous and crystalline forms of A-10. The data suggests that glutamic acids are in identical environments in the crystalline and amorphous samples. The fast relaxation rates of their alpha, beta and gamma carbons suggest that glutamic acid residues are excluded from the crystalline regions in both the samples. Static solid state NMR experiments have been carried out on oriented mats of crystalline A-10. The orientation of the crystallites has been studied by the analysis of lineshapes. The lineshape analysis is consistent with the crystallites being an imperfect form of an orientation in which the crystallographic b axis (Figure 1.6) is perpendicular to the mat and there is free rotation around b. (Abstract shortened by UMI.)
Bibliographical Information: