Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein

Antifreeze proteins are prevalent in many species that experience freezing or close to freezing temperatures. The proteins lower the freezing point of water, enabling the species to survive subzero temperatures. One of these species is fish, in which antifreeze proteins originally were discovered. While antifreeze proteins can be found in a wide variety of secondary and tertiary structures, the α helical antifreeze protein from winter flounder (WflAFP) is probably the most extensively studied. The antifreeze activity of all antifreeze proteins has been correlated to size and flatness of the ice-binding surface which for WflAFP has been demonstrated to be on one side of the alpha-helix, comprising primarily regularly spaced alanine and threonine side chains. The analysis of the size necessary for activity, however, was complicated in the case of WflAFP as a shortening of the sequence also lead to a reduction in α helicity. To overcome this difficulty, in this work a C-terminal stabilizing and alpha-helicity inducing capping unit, the Trp-cage, was employed. In a first step, the effect of the C-terminal modification on the wild-type antifreeze protein had to be studied to eliminate any interference of the modification on ice activity. In a second step, after eliminating any negative influence of the capping unit, the dependence of ice activity on the size of the ice-binding surface could be analyzed. The combination of both Trp-cage and antifreeze protein segment was pursued using two different approaches: The fusion approach was based on the synthesis of both the Trp-cage capping unit and an antifreeze protein segment separately and fusing them together in a second step. The chimera approach is based on the fact, that in the primary sequence of both Trp-cage and antifreeze protein some amino acids are interchangeable leading to an overlapping sequence and overall a shorter protein. Four alpha-helical antifreeze protein variants of different lengths were stabilized using the Trp-cage. Of the four ice-binding Trp-cage chimera (IBTC), three were also obtained as GFP labeled variants. All IBTC were characterized using NMR , CD , and UV/vis spectroscopy, mass spectrometry and their antifreeze activity or ice growth retardation as well as ice shaping abilities were determined. All ice-binding Trp-cage chimera designed based on the chimera approach were well folded. The IBTC designed based on the fusion approach, on the other hand, had a less pronounced fold according to 1H NMR data. All folded IBTC also satisfied the criteria for α helicity in a chemical shift deviation plot and the CD data also indicate α helicity for all IBTC. The analysis of ice activity gave surprising results. The IBTC based on the wild-type antifreeze protein had comparable antifreeze activity to WflAFP. However, no antifreeze activity could be observed for the other IBTC. As a result, even though the C-terminal capping unit has no influence on the antifreeze activity of the IBTC, the induction and stabilization of α helicity is not the only criteria necessary for activity. Rather, the size of the ice-binding surface has to match the one present in nature to generate antifreeze activity.


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