Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

Abstract

BackgroundIce nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown.ResultsHere we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins.ConclusionsDimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

“…The { 1 H- 15 N} heteronuclear single quantum coherence (HSQC) spectra were acquired with the 1 H carrier centered at the water frequency (5.01 ppm) and recorded with spectral widths of 6000 Hz ( 1 H) and 1400 Hz ( 15 N), respectively. The chemical shift was referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), and the temperature calibration of the probe was performed by monitoring the 1 H chemical shifts of methanol.…”

Immersion freezing of ice nucleation active protein complexes

“…Larger protein oligomers are considered to be active at higher temperatures and are thought to be related to class I and II bacterial IN (Schmid et al, 1997). Up to now, the structure and functionality of the INA protein oligomers has not been finally clarified and is still the object of contemporary research (Garnham et al, 2011).…”

“…It is concluded that oligomers consisting of two up to a few single proteins could correspond to class III IN, i.e., initiate freezing in the temperature range from about −7 • C to −10 • C (Govindarajan and Lindow, 1988;Garnham et al, 2011), where the majority of the INA bacteria can be ice nucleation active. Larger protein oligomers are considered to be active at higher temperatures and are thought to be related to class I and II bacterial IN (Schmid et al, 1997).…”

“…Southworth et al (1988) reported that INA proteins were found in groups of two to three. Through use of γ -radiation analysis, i.e., by gradually decreasing biological activity in a P. syringae sample, Govindarajan and Lindow (1988) derived that a single INA protein induces freezing at −12 • C. Recently, Garnham et al (2011) proposed that the INA proteins naturally form…”

“…Thereby, different bacterial species and strains may feature different patterns of gene expression (for an overview see Després et al, 2012). The apparent molecular weight of an INA protein was estimated to be 120-180 kDa (Kawahara, 2002) and its active surface area was given by Garnham et al (2011) as 4 nm × 32 nm.…”