Question

select 2 amino acid residues that form hydrogen bonds with your ligand and using the mutagenesis wizard menu, carry out a site-directed mutagenesis of these 2 residues. that is, replace them with 2 other amino acids in your primary protein sequence on pymol. you must make sure to replace these 2 amino acids with ones that have different properties (eg. if a hydrophilic amino acid is in the original primary sequence, replace it with a hydrophobic amino acid, if a charged amino acid is in the sequence, replace it with an uncharged amino acid etc). from the amino acids that you identified in question 3 above, which are you replacing and why? report the names and properties of the amino acids before and after you replace them.

Answer 1

Site-directed mutagenesis can be conducted by replacing amino acids in a protein that interact with a ligand through hydrogen bonds. For example, replacing histidine (a polar, positively charged amino acid) with phenylalanine (a nonpolar, neutral amino acid) would significantly alter the ligand interaction.

Step-by-step explanation:

To perform site-directed mutagenesis on a protein interacting with a ligand, we first need to identify the amino acid residues forming hydrogen bonds with the ligand. If we consider the example provided where His12 and His119 are involved in hydrogen bonding with a ligand, we could choose to replace one or both of these with amino acids that have different properties.

For instance, histidine (His) is a polar, positively charged amino acid under physiological conditions. We might replace His with an amino acid like phenylalanine (Phe), which is nonpolar and neutral. This would alter the interaction with the ligand because Phe cannot form the same hydrogen bonds as His. Similarly, if we consider another residue like Arg39 (which is positively charged and polar because of its guanidinium group), we could replace it with an amino acid like leucine (Leu), which is hydrophobic and neutral. These substitutions would have a significant impact on the ligand binding due to the different properties of the new amino acids replacing the original ones.