The objective of this research project is to engineer new protein frameworks for attaching biophysically or biologically active molecules in a geometrically defined and synthetically controlled spacing. We shall rationally design, chemically synthesize, and structurally and functionally characterize new betabellins, a class of beta-sheet proteins. These polypeptide frameworks will contain useful structural features that have rarely or never been seen in natural proteins, such as type-I' beta turns, D-amino acids, and new proline-based amino acids. They are called """"""""proteins"""""""" because they mainly contain the standard genetically encoded alpha-amino acids and because they fold into a single discrete molecular structure in aqueous solution. They are called """"""""nongenetic"""""""" proteins because in most cases they cannot be prepared like the """"""""genetic"""""""" proteins of biology through gene-encoded biosynthesis on ribosomes. The overall process is called """"""""engineering"""""""" because it proceeds in three stages. First, rational protein design involves computer-aided molecular modeling of the betabellin target structure by using structural insights, molecular graphics, molecular mechanics, and molecular dynamics simulations. Second, modular protein synthesis involves chemical assembly of protected amino acids by the solid-phase method, chromatographic purification of the target molecule to homogeneity, and determining its molecular mass by electrospray- ionization mass spectrometry, molecular composition by amino acid analysis, and amino acid sequence by Edman degradation. Third, protein characterization includes assessing relative solubility by liquid-liquid partitioning, hydrophilicity by reversed-phase high-pressure liquid chromatography, the presence and stability of alpha helices or beta sheets by circular dichroic spectroscopy, and three-dimensional structure in solution by two-dimensional nuclear magnetic resonance spectrometry or in the crystal by X-ray crystallography. Engineering of novel betabellins will involve designing, synthesizing, and characterizing of betabellins that explore the roles of beta turns, loops, disulfide bonds, hydrophobic surfaces, and metal ion-binding sites in protein folding. These results will be very useful as a guide for biotechnologists to genetically engineer novel protein structures for diagnosis and therapy.
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