Transcript Managing people in sport organisations: A strategic human

FIGURE 11.1
Introduction of Disulfide Bonds
A disulfide bond can be added to a protein by changing two amino acids into cysteines by site-directed
mutagenesis. When the engineered protein is put under oxidizing conditions, the two cysteines form a
disulfide bond, holding the protein together at that site.
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Results for disulfide additions to T4
Lysozyme
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Alpha Helix Dipole
How can we stabilize a protein? We can “cap” alpha helices that are
not naturally caped for stability all
ready.
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FIGURE 11.3
Difference in Structure between NAD+ and NADP+
NAD (nicotinamide adenine dinucleotide) differs from NADP by one single phosphate (yellow) which
will deprotonate at pH = 7.
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FIGURE 11.4
Changing Cofactor Preference of Lactate Dehydrogenase
Lactate dehydrogenase (LDH) preferentially binds NAD because the binding pocket has an aspartic
acid. The negatively charged carboxyl repels the negatively charged phosphate of NADP. Changing
the aspartic acid to serine allows either NAD or NADP bind to LDH. Adding a positively charged lysine
makes the pocket more attractive to the NADP.
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FIGURE 11.5
Scaffold Minimization
Proteins often have large structural domains (orange) outside the active site (blue/red). These
domains can be engineered to make them smaller (which often enhances the proteins’ level of
expression) or to make isolation and purification easier.
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FIGURE 11.6
Adding New Functional Groups to Proteins
(A) Nonnatural amino acids add new functional groups. These can be incorporated into a protein
during translation. (B) The nonnatural amino acid, pBpa, crosslinks the GST mutant protein to form a
homodimer.
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