Protein Engineering Protocols - Mycobacteriology research center

162 Bradley et al.two of these proteins were determined by NMR and shown to be well-orderedfour-helix bundles, as specified by design (12,31).2.2. Codon UsageAs mentioned in Heading 1., the degenerate codons NAN and NTN encodepolar and nonpolar amino acids, respectively (Fig. 2). However, simply usingan equimolar mixture of A, C, G, and T at the combinatorial N positions wouldintroduce undesirable traits into the sequence. Most importantly, an unconstrainedNAN codon would encode an unacceptably high frequency of stopcodons (i.e., 2 out of 16 NAN codons 12.5%). Below is a list of considerationswe have used in the design of polar and nonpolar codons.2.2.1. The NAN (Polar) Codon1. At the first base of the NAN codon, we use an equimolar mixture of G, C, and A.T is excluded, thereby eliminating the possibility of stop codons and tyrosine (seeNote 3). If all bases were included at this first position, then the presence ofthymine would give rise to the stop codons TAG or TAA.2. The mixture of nucleotides at the third base of the NAN codon can be altered to favorsome amino acids over others. An equimolar mixture of G, C, A, and T would yieldan equal likelihood of histidine, glutamine, asparagine, lysine, aspartate, and glutamate.However, some of the residues of the NAN codon have higher intrinsic propensitiesthan others to form α-helices (32–35). By omitting T at the third position, wecan favor glutamine, lysine, and glutamate over histidine, asparagine, and aspartate.This matches theses residue’s intrinsic propensities to form α-helices (32–35).2.2.2. The NTN (Nonpolar) CodonEquimolar mixtures of all four bases at the first and third N positions of theNTN codon would encode six times as many leucines as methionines (i.e., sixleucine codons vs one methionine codon). In addition, an equimolar mixturewould encode protein sequences in which a quarter of the hydrophobic residues inthe interiors would be valine. Because valine has a relatively low α-helical propensity,this may be undesirable for some designs. By altering the molar ratio of themixture at the first and third N positions, the relative abundance of hydrophobicresidues can be altered. For example, in the initial four-helix bundle library, thefirst base of the NTN codon contained A:T:C:G in a molar ratio of 3:3:3:1 and thethird base mixture contained equimolar concentrations of G and C (8). By biasingthe mixture in this way, valine is limited to only 10% of the hydrophobic residuesand leucine is represented only three times as frequently as methionine.2.2.3. Codon Usage of the Host Expression SystemThe DNA sequences in both the constant and the combinatorial regions ofthe library should be biased to favor those codons used most frequently in the