Water-organizing motif continuity is critical for potent ice nucleation protein activity

Abstract

Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section’s deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Insect Freeze-Tolerance Downunder: The Microbial Connection

“…There are many possible sources of ice+ agents in the environment and within the insect, of different sizes, stability, and efficiency. For example: ( A ) manufactured by the insect, e.g., lipoproteins in Tipula trivittata [ 46 ], ( B ) derived from microbes, e.g., large proteins from Pseudomonas borealis [ 47 ], ( C ) created by plants, e.g., class II chitinase [ 48 ], ( D ) environmental particles, e.g., kaolin-iron oxide composites [ 49 ], and ( E ) manufactured by fungi, e.g., fungal hydrophobins [ 50 ].…”

Preferential Ordering and Organization of Hydration Water Favor Nucleation of Ice by Ice-Nucleating Proteins over Antifreeze Proteins

“…The truncated PbINP (residues 894−1025) containing 8 repeat units was obtained by using the predicted β-helical fold for the INP (P. borealis) by Peter L Davies group. 29 The repetitive domain, modeled by AlphaFold is the continuous βsolenoid in which each 16 residue repeat forms one coil of the solenoid. There are 65 such repeats along with a 41 residue Cterminal domain and a 110 residue N-terminal domain.…”

Poly(vinyl alcohol) Molecular Bottlebrushes Nucleate Ice

“…The study of INPs is challenging due to the low solubility of the INP and the need for the transmembrane/lipid anchored domains for activity. Davies and co-workers reported recombinant mutated INPs, but it was essential to study activity while attached to the host organism (E.coli) to obtain ice nucleation functional data …”

“…Davies and co-workers reported recombinant mutated INPs, but it was essential to study activity while attached to the host organism (E.coli) to obtain ice nucleation functional data. 5 In addition to IBPs, it has emerged that synthetic materials can also bind ice, reproducing the macroscopic properties of IBPs�presenting opportunities not only for biomedical applications but also to understand IBP function. 6−8 Synthetic polymers can be produced with excellent control over architecture and composition, allowing molecular shape and composition to be tuned to probe the material/ice interface.…”

“…Davies and co-workers reported recombinant mutated INPs, but it was essential to study activity while attached to the host organism ( E.coli ) to obtain ice nucleation functional data. 5 …”