Backbone hydrogen bonding interaction of the inactive isoform of type III antifreeze proteins studied by 1H/15N-HSQC spectra

Antifreeze proteins (AFPs) bind to the ice crystals and then are able to inhibit the freezing of body fluid at subzero temperatures. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We prepared the QAE2 (AFP11) and SP (AFP6) isoforms of the notched-fin eelpout AFP and their mutant constructs and determined their temperature gradients of amide proton chemical shifts (AS/AT) using NMR. The nfeAFP11 (QAE2) has the distinct AS/AT pattern of the first 310 helix compared to the QAE1 isoforms. The nfeAFP6 (SP) has the deviated AS/AT values of many residues, indicating its backbone conformational distortion. The study suggests the distortion in the H-bonding interactions and backbone conformation that is important for TH activities.