Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein

Abstract

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10Ϫ30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive β structure (70Ϫ80 %) with little or no A helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0°C to 20°C), and the presence of 50% trifluoroethanol.Keywords : CD; disulfide linkages; NMR; protein folding; thermal hysteresis.Antifreeze proteins (AFPs), traditionally called thermal hysteresis proteins (THPs) in the insect literature, have recently been purified and characterized from two unrelated insect species, spruce budworm (Choristoneura fumiferana), and the common yellow mealworm (Tenebrio molitor) [1,2]. Their ability to depress the non-equilibrium freezing point of a solution below its melting point (thermal hysteresis activity) is exceptional in two respects. One is that the freezing point depression (4Ϫ5°C) measured in these insects is several degrees greater than that seen with AFPs from other organisms such as fish and plants [3,4]. The other is that their specific thermal hysteresis activity (freezing point depression as a function of concentration) is at least an order of magnitude greater than that obtained with fish AFPs [5,6]. Despite their superior activity, insect THPs operate at the ice/water interface to control ice crystal growth and shape and, therefore, appear to function by the adsorption-inhibition mechanism originally described for fish AFPs [7].The recently described spruce budworm (moth) and mealworm (beetle) THPs are present in larval hemolymph. Both are small, Cys-, Thr-, and Ser-rich proteins of 8Ϫ9 kDa that are extensively disulfide bonded. Despite these physical resemblances and their similar hyperactivity, they show no obvious homology at the DNA or amino acid sequence level, and the spacing of their numerous cysteine residues is quite different. Moreover, the mealworm THP is clearly built up of 12-aminoacid repeats, wherea...

Folding and Structural Characterization of Highly Disulfide-Bonded Beetle Antifreeze Protein Produced in Bacteria

“…It is noteworthy that the misfolded TmTHP does not contain free -SH groups and that a proportion of misfolded species can be converted to the folded form by gentle heating and cooling, which would not disrupt the arrangement of the disulfide bonds. A similar observation has been made with spruce budworm THP, which is a 9.0-kDa protein that is less extensively disulfide bonded (4 instead of 8) in a distinctly different pattern (12). In each case the heat treatment leads to an appreciable increase in yield.…”

“…To check for the presence of TmTHP in inclusion bodies, the pellet was resuspended in 8 ml of 8 M guanidine-HCl, 100 mM Tris-HCl (pH 8.5), 1 mM EDTA, 12 mM 2-mercaptoethanol and gently mixed at room temperature (22°C) for 1 h. This suspension was added dropwise to 160 ml of ice-cold refolding buffer, 50 mM K 2 HPO 4 , 1 mM EDTA, 2 mM 2-mercaptoethanol, and 100 mM NaCl (pH 10.7), and left to stir overnight at 15°C. The solution was dialyzed over 2 days against 10 mM Tris-HCl, 1 mM EDTA, and 100 mM NaCl, pH 8.0 (12), and left to oxidize for up to 1 month.…”

A β-Helical Antifreeze Protein Isoform with Increased Activity

“…AFP expressed in BL21(DE3) cells was present in inclusion bodies requiring refolding performed in a manner similar to that of CfAFP-337 (14). Refolded protein was purified using fast protein liquid chromatography and high pressure liquid chromatography column chromatography as reported for CfAFP-337 (14) with the exception that the majority of the CfAFP-501 protein eluted in a single, well folded peak from both the fast protein liquid chromatography and high pressure liquid chromatography columns without the splitting into incompletely folded and well folded peaks as seen with CfAFP-337.…”

Characterization of antifreeze protein gene expression in summer spruce budworm larvae

“…3). Conserved Cys residues in long isoforms include the disulfide-bonded pairs Cys4 & 17, Cys25 & 37, Cys55 & 67, Cys92 & 115 and Cys97 & 110 (Leinala et al, 2002a;Gauthier et al, 1998). Two Cys residues, which presumably contribute to protein stability, were absent in the CfAFP6 isoform with substitutions of Arg and Tyr at positions 25 and 55, respectively (Fig.…”