Abstract
Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.
Highlights
Sub-zero temperatures are fatal in most organisms by kinetically slowing down vital biochemical reactions, denaturating biomolecules, or rupturing cell membranes
Studies over several decades have revealed that antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) act as biological inhibitors of ice crystal formation by depressing the water freezing point in a non-colligative manner [2,3], a process known as thermal hysteresis (TH) [4]
Over the past half century, more AFPs have been isolated from different organisms and are classified into four major types: (1) type I AFPs are described as having Ala-rich protein sequences with amphipathic a-helical structures and varying sizes between 3.3 kDa and 4.5 kDa [7,8,9,10]; (2) type II AFPs are larger, globular folded proteins with multi-Cys residues bridged by disulphide bonds [11,12,13]; (3) type III AFPs are described as globular proteins with molecular weights of approximately 6 kDa [14,15,16,17]; and (4) type IV AFPs are a-helical in structure with multi-Glu (E) or Gln (Q) residues in their sequences [18]
Summary
Sub-zero temperatures are fatal in most organisms by kinetically slowing down vital biochemical reactions, denaturating biomolecules, or rupturing cell membranes. In agreement with Darwin’s theory of natural selection, Antarctic and Arctic organisms, including plants, animals, fungi and bacteria, have developed a unique adaptive mechanism of survival by producing antifreeze proteins (AFPs) and antifreeze glycopeptides (AFGPs) [1]. Studies over several decades have revealed that AFPs and AFGPs act as biological inhibitors of ice crystal formation by depressing the water freezing point in a non-colligative manner [2,3], a process known as thermal hysteresis (TH) [4]. The first AFP was discovered in the blood of Antarctic fish over 40 years ago [5,6]. Type V AFPs have been reported from insects and are known as hyperactive proteins [19]
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