Interactions of β-Helical Antifreeze Protein Mutants with Ice

The fold of the β-helical antifreeze protein from Tenebrio molitor (TmAFP) proved to be surprisingly tolerant of multiple amino acid substitutions, enabling the construction of a panel of mutants displaying grids of single amino acid types in place of the threonines on the ice-binding face. These mutants, maintaining the regularity of amino acid spacing found in the wild-type protein but with different functional groups on the surface, were tested for antifreeze activity by measuring thermal hysteresis and observing ice grown in their presence. We found that no mutant exhibited the dramatic activity of the wild-type version of this hyperactive antifreeze protein. However, mutants containing four valines or tyrosines in place of the threonines in the center of the TmAFP ice-binding face showed residual thermal hysteresis activity and had marked effects on ice crystal morphology. The results are discussed in the context of a two-stage model for the absorption−inhibition mechanism of antifreeze protein binding to ice surfaces

Modeling the Influence of Antifreeze Proteins on Three-Dimensional Ice Crystal Melt Shapes using a Geometric Approach

The melting of pure axisymmetric ice crystals has been described previously by us within the framework of so-called geometric crystal growth. Nonequilibrium ice crystal shapes evolving in the presence of hyperactive antifreeze proteins (hypAFPs) are experimentally observed to assume ellipsoidal geometries ("lemon" or "rice" shapes). To analyze such shapes we harness the underlying symmetry of hexagonal ice Ih and extend two-dimensional geometric models to three-dimensions to reproduce the experimental dissolution process. The geometrical model developed will be useful as a quantitative test of the mechanisms of interaction between hypAFPs and ice.Comment: 15 pages, 5 figures; Proc. R. Soc. A, Published online before print June 27, 201

Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 18C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH

New insights into ice growth and melting modifications by antifreeze proteins

Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs

Freezing and Melting Hysteresis Measurements in Solutions of Hyperactive Antifreeze Protein from an Antarctic Bacteria

Antifreeze proteins (AFPs) evolved in cold-adapted organisms and serve to protect them against freezing in cold conditions by arresting ice crystal growth. Recently, we have shown quantitatively that adsorption of AFPs not only prevents ice from growing but also from melting. This melting inhibition by AFPs, which results in superheated ice (Celik et al, PNAS 2010), is not a well-known phenomenon. Here we present our recent findings in which the Ca2+-dependent hyperactive AFP from Marinomonas primoryensis (MpAFP) clearly displays this property. Additionally, we found that an ice crystal that is initially stabilized and protected by this type of AFP can be overgrown and then melted back to the original crystal. This repeatable process is likely due to melting inhibition, and supports the idea that AFPs bind irreversibly to ice surfaces

Structural Basis for the Superior Activity of the Large Isoform of Snow Flea Antifreeze Protein

The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules

Adaptation and psychometric testing of the Turkish evaluation of daily activity questionnaire in people with rheumatoid arthritis

Purpose: The aims were to translate the Evaluation of Daily Activity Questionnaire (EDAQ) into Turkish, then test validity and reliability in people with rheumatoid arthritis (RA) in Turkey. Material and Methods: Phase 1: The EDAQ was forward and backward translated, culturally adapted following cognitive debriefing interviews with participants with RA (n=10) and finalised by an expert committee. Phase 2: Participants (n=215) completed a questionnaire including the EDAQ, Health Assessment Questionnaire (HAQ), and Short-Form 36 v1 (SF-36v1). Two weeks later, the EDAQ was again completed for test-retest reliability (n=82:38%). Internal construct validity was assessed using Rasch analysis. Internal consistency, concurrent validity, and test-retest reliability were assessed. Results: Following cultural adaptation, one item was removed, and examples increased or changed. Cronbach’s α values were 0.71 – 0.93 for all EDAQ domains, i.e., acceptable to good. The EDAQ met Rasch model requirements for fit (excellent construct validity: p>0.05). Concurrent validity was moderate to strong for most EDAQ domains with HAQ (rs 0.49-0.81) and SF-36-v1 Physical Function (rs 0.42-0.70). There was excellent test-retest reliability for all domains (ICC (2,1): 0.95-1.00).Conclusion: The Turkish EDAQ is a valid, reliable measure of daily activity ability for use in practice and research with Turkish speakers with RA

CPAP Intervention as an Add-On Treatment to Lipid-Lowering Medication in Coronary Artery Disease Patients with Obstructive Sleep Apnea in the RICCADSA Trial

Dyslipidaemia is a well-known risk factor for coronary artery disease (CAD), and reducing lipid levels is essential for secondary prevention in management of these high-risk individuals. Dyslip-idaemia is common also in patients with obstructive sleep apnea (OSA). Continuous positive airway pressure (CPAP) is the first line treatment of OSA. However, evidence of a possible lipid-lowering effect of CPAP in CAD patients with OSA is scarce. We addressed the effect of CPAP as an add-on treatment to lipid-lowering medication in a CAD cohort with concomitant OSA. This study was a secondary analysis of the RICCADSA trial (Trial Registry: ClinicalTrials.gov; No: NCT 00519597), that was conducted in Sweden between 2005 and 2013. In total, 244 revascularized CAD patients with nonsleepy OSA (apnea–hypopnea index ≥ 15/h, Epworth Sleepiness Scale score < 10) were randomly assigned to CPAP or no-CPAP. Circulating triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels (all in mg/dL) were measured at baseline and 12 months after randomization. The desired TG levels were defined as circulating TG < 150 mg/dL, and LDL levels were targeted as <70 mg/dL according to the recent guidelines of the European Cardiology Society and the European Atherosclerosis Society. A total of 196 patients with available blood samples at baseline and 12-month follow-up were included (94 randomized to CPAP, 102 to no-CPAP). We found no significant between-group differences in circulating levels of TG, TC, HDL and LDL at baseline and after 12 months as well as in the amount of change from baseline. However, there was a significant decline regarding the proportion of patients with the desired TG levels from 87.2% to 77.2% in the CPAP group (p = 0.022), whereas there was an increase from 84.3% to 88.2% in the no-CPAP group (n.s.). The desired LDL levels remained low after 12 months in both groups (15.1% vs. 17.2% in CPAP group, and 20.8% vs. 18.8% in no-CPAP group; n.s.). In a multiple linear regression model, the increase in the TG levels was predicted by the increase in body-mass-index (β = 4.1; 95% confidence interval (1.0–7.1); p = 0.009) adjusted for age, sex and CPAP usage (hours/night). CPAP had no lipid-lowering effect in this revascularized cohort with OSA. An increase in body-mass-index predicted the increase in TG levels after 12 months, suggesting that lifestyle modifications should be given priority in adults with CAD and OSA, regardless of CPAP treatment