Protein Engineering Protocols - Mycobacteriology research center

306 IndexCCalcium indicators, see ChameleonsCalmodulin, see ChameleonsCD, see Circular dichroismChameleons,advantages and limitations, 71, 80calcium-binding curve generation,76, 77, 81cloning, 75, 81design, 72–74, 80fluorescence resonance energytransfer principles, 72fluorescence spectroscopycharacterization, 76imaging,cell culture, 78data acquisition, 78–81materials for preparation andimaging, 74, 75, 80, 81purification, 75, 76, 81χ 2 test, experimental bias testing oflibraries, 148, 149, 152Circular dichroism (CD), zinc fingerproteins, 87Coiled coil,design,computational protein design,59–61, 64helix orientation,edge residues, 50, 51, 63nonpolar core residues, 49,50, 63polar core residues, 50, 63overview, 37, 38pairing specificity,edge residues, 47–49, 61–63nonpolar core residues, 45,46, 62polar core residues, 46, 47, 62quaternary structure,edge residues, 44, 45, 62nonpolar core residues, 38–42, 61polar core residues, 42–44, 61selection,codon bias, 56, 57degenerate codons, 55, 56overview, 55phage display, 59protein fragmentcomplementation assay,58, 59repressor assay, 59yeast two-hybrid system, 59semi-rational design, 55stability,helical length, 52, 64helix capping, 54, 64helix dipole interactions, 53,54, 61, 64helix propensity, 52, 53solubility considerations, 51, 52peptide velcro hypothesis, 37, 61structures, 35–37Combinatorial libraries,binary patterning of polar andnonpolar amino acids, seeBinary patterningde novo protein design, 3, 4, 7directed protein design, seeComputational protein designDNA shuffling, see Degenerateoligonucleotide gene shuffling;Nucleotide exchange andexcision technologygene libraries for protein profiles,15–18monobody construction,double-stranded DNA synthesis,101, 108electrocompetent cell preparation,101, 102, 108electroporation for phagemidDNA, 102mutagenic oligonucleotidepreparation, 101, 108uracil single-stranded phagepreparation, 100, 101yeast library construction, 102, 103