Increased flexibility decreases antifreeze protein activity

Abstract

Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single a-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg 37 HPLC6, nonamidated HPLC6 Ala 37 , amidated HPLC6 Ala 37 , and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still a-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala 37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.

Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive

“…The extra alanines, up to 10 in a row, take the place of Asn and Leu that are not thought to play a critical role in either structure or function of type I AFP. The N-terminal D 1 TASD sequence and C-terminal amidation that form the α-helix capping structures are well conserved 17 25 .…”

Troubleshooting Guide to Expressing Intrinsically Disordered Proteins for Use in NMR Experiments

“…The two advantages of this tag are that the cleavage is carried out by a highly specific SUMO-protease, which recognizes the entire SUMO domain, rather than just a short recognition sequence, and the other advantage is that the protocol leaves a “native” (as in user-defined) N-terminus on the IDP. While commercial sources for the SUMO protease are available, we have found it cost efficient to produce our own (Reverter and Lima, 2009; Patel and Graether, 2010).…”

Ice Binding Proteins: Diverse Biological Roles and Applications in Different Types of Industry

“…DeVries' hypothesis was levered by mutagenesis of two and four centrally placed threonines to serines, because AFPs' activity lowered to 0-10% (in comparison to the wild type AFPs). On the other hand, when threonine residues were exchanged to valines, the loss of activity dropped to about 15% [95]. This fact brought the new hypothesis of the hydrophobic effect, which assumed that molecules of water located between protein and ice are pushed out to bulk solvent and AFPs bonding to ice is created because of high entropy.…”