【文章內容簡介】 utants: ++11 ?22176。C Disulfide bridges increase protein stability (contd.) Glycine and proline have opposite effects on stability A glycine residue at a specific position in a protein has usually one conformation in a folded structure but can have many different conformations in different unfolded structures of the same protein and thereby contribute to the diversity of unfolded conformations. Proline residues have less conformational freedom in unfolded structures than any other residue Glycine and proline have opposite effects on stability (contd.) Another way to decrease the number of possible unfolded structures of a protein is to mutate Gly residues to any other residue or to increase the number of Pro residues. Results: in T4 lysozyme, Gly77Ala (Tm increase of 1176。 C) Ala82Pro (Tm increase of 2176。 C) Glycine and proline have opposite effects on stability (contd.) The threedimensional structures of these mutant enzymes were also determined: the Ala82Pro mutant had a structure essentially identical to the wildtype except for the side chain of residue 82。 this strongly indicates that the effect on Tm of Ala82Pro is indeed due to entropy changes. Stabilizing the dipoles of ? helices increases stability The helix dipole concept: the positive charge is at the Nterminus of the helix, and the negative charge is at the Cterminus of the helix. Thus, negative ions are usually bound to the Nterminal end of the helix. Results, in T4 lysozyme, Ser38Asp (Tm increase of 2176。 C) Asn144Asp (Tm increase of 2176。 C) Stabilizing the dipoles of ? helices increases stability (contd.) Stabilizing the dipoles of ? helices increases stability (contd.) Mutations that fill cavities in hydrophobic cores do not stabilize T4 lysozyme, although they may stabilize other proteins The elimination of a cavity of the size of one –CH2 group in the protein interior stabilizes the enzyme by about 1 kcal/mol. Results: in T4 lysozyme, Leu23Phe amp。 Ala129Val: these new side chains were hydrophobic and large enough, in principle, to fill the cavities without making too close contacts with surrounding atoms. Mutations that fill cavities in hydrophobic cores do not stabilize T4 lysozyme, although they may stabilize other proteins (contd.) In practice, they were both less stable than wildtype by to kcal/mol. It turns out that in order to fill the cavities, the new side chains in the mutants adopt energetically unfavorable conformations. This introduces strain in the structure. Mutations designed to fill existing cavities may be effective in some cases, but they are not likely to provide a general route to substantial improvement in protein stability. Proteins can be engineered by binatorial methods Combinatorial methods: also called in vitro or directed evolution techniques Combinatorial methods: in which, libraries of related proteins are analyzed simultaneously. By sorting these libraries to select for a particular function, the small number of active proteins can be separated from millions of inactive variants. Proteins can be engineered by binatorial methods (contd.) Methods of generating binatorial DNA libraries: (a) oligonucleotidedirected mutagenesis, (b) errorprone