1. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 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. FIGURE 11.1

2. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 2 Results for disulfide additions to T4 Lysozyme

3. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 3 Alpha Helix Dipole How can we stabilize a protein? - We can “cap” alpha helices that are not naturally caped for stability all ready.

4. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 4 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. FIGURE 11.3

5. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 5 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. FIGURE 11.4

6. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 6 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. FIGURE 11.5

7. Companion site for Biotechnology. by Clark Copyright © 2009 by Academic Press. All rights reserved. 7 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. FIGURE 11.6