Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

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.

Will It Be Beneficial To Simulate the Antifreeze Proteins at Ice Freezing Condition or at Lower Temperature?

“…Note that the antifreeze property of peptide 1m is approximately 50-to 100-fold less compared to typical AFPs. 16 On the contrary, similar trend is observed for control peptide (LL37) at all temperature conditions, where any deviation is attributed to temperature difference over the complete system and is not attributed to functional aspect of protein. Typically, the PCA projections for LL37 remain unchanged for 300 and 273 K. This is reflective with realistic projection pattern ( Figure 1E), which is indicative of attenuation for sufficient dynamicity.…”

“…An example is present in our previous report, where AFPs have been engineered from globular protein G. antarctica correlating the antifreeze activity with the helicity and geometrical straightforwardness. 16 In general, the details of biophysical and biochemical properties along with structural studies are usually described using relevant methodologies and techniques. For instance, the structural inference and antifreeze activity conclusion include results from NMR, X-ray, ice recrystallization inhibition assay, and thermal hysteresis.…”

“…19 Peptide 1m, on the other hand, is a short 25-residue peptide engineered from a globular folded AFP. 16 Notably, these AFPs have a varying range of potency in terms of antifreeze activity, which will be helpful in quantifying the successive results. The dynamic properties of antifreeze peptide were investigated at room temperature (300 K) and at lower freezing conditions of 273 and 227 K.…”

“…23−25 For comparison purposes, we have used the nonantifreeze protein LL37 as a control. 16 To the best of our knowledge, a comparative study of nonantifreeze protein using TIP4P/Ice model with typical AFPs has not been reported in the literature.…”

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

“…Amino-acid sequence analysis showed that Afp1 contains four α-helices. Shah et al confirmed the antifreeze activity of each helical peptide [22]. In addition, the NMR structures of the peptides were determined and the ice-binding model was generated using a molecular dynamics method.…”