The binding affinity between two specific protein molecules can change under varying pH conditions due to several factors, including changes in the ionization state of amino acid residues, alterations in protein conformation, and the influence of pH on the overall electrostatic interactions between the proteins. Here, we will discuss these factors in detail and provide experimental evidence to support these claims.1. Changes in the ionization state of amino acid residues:The ionization state of amino acid residues in proteins is highly dependent on the pH of the surrounding environment. At different pH values, the protonation and deprotonation of acidic Asp, Glu and basic Lys, Arg, His amino acid residues can occur, leading to changes in the overall charge of the protein. This change in charge can affect the electrostatic interactions between the two protein molecules, thereby altering their binding affinity.Experimental evidence: A study by Schreiber and Fersht 1993 investigated the pH dependence of the binding affinity between barnase and barstar, two proteins that form a tight complex. They found that the binding affinity decreased significantly at both low and high pH values, which they attributed to the protonation and deprotonation of specific amino acid residues involved in the binding interface.2. Alterations in protein conformation:Changes in pH can also lead to alterations in the conformation of proteins, which can affect their binding affinity. This can occur due to the protonation or deprotonation of amino acid residues, which can disrupt hydrogen bonding networks and cause changes in the overall protein structure. Additionally, changes in pH can lead to the exposure or burial of hydrophobic regions, which can also impact protein-protein interactions.Experimental evidence: A study by Krezel and Bal 2018 examined the pH-dependent conformational changes in calmodulin, a calcium-binding protein. They found that at low pH, calmodulin adopts a more compact conformation, which reduces its ability to bind target proteins.3. Influence of pH on electrostatic interactions:The overall electrostatic interactions between two protein molecules can be influenced by the pH of the surrounding environment. At different pH values, the protonation and deprotonation of amino acid residues can lead to changes in the overall charge distribution of the proteins, which can affect the strength and direction of electrostatic interactions between them.Experimental evidence: A study by Zhou et al. 2008 investigated the pH-dependent binding affinity between the proteins p53 and MDM2. They found that the binding affinity decreased at low pH, which they attributed to the protonation of histidine residues in the p53 binding site, leading to a reduction in electrostatic interactions with MDM2.In conclusion, the binding affinity between two specific protein molecules can change under varying pH conditions due to changes in the ionization state of amino acid residues, alterations in protein conformation, and the influence of pH on the overall electrostatic interactions between the proteins. Understanding these factors can help researchers design better drugs and therapeutic strategies that target specific protein-protein interactions.