Vettai S. Ananthanarayanan

Structural studies on the freezing-point-depressing protein of the winter flounder Pseudopleuronectes americanus.

Abstract The freezing-point-depressing protein from the winter flounder, Pseudopleuronectes americanus has been shown from circular dichroism measurements to possess a large proportion (∼85%) of the α-helical conformation in aqueous solution (pH 8.0) at −1°C. The helical content decreases as the temperature is raised. Viscosity data at −1°C indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this freezing-point-depressing protein as well as its primary structure (reported elsewhere), are found to be different from its counterpart glycoproteins isolated from the Antarctic fish.

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

SummaryEight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.

Antifreeze proteins from the ocean pout, Macrozoarces americanus: circular dichroism spectral studies on the native and denatured states

Abstract CD spectral data on the antifreeze proteins from the ocean pout ( Macrozoarces americanus ) have been obtained under native and denaturing conditions. At low temperatures (near 0°C), the far-ultraviolet CD spectra of the two known immunologically distinct groups of ocean pout proteins are found to be qualitatively similar to those of the antifreeze glycoproteins, with a distinct positive band near 225 nm. However, unlike the latter, the ocean pout proteins exhibit CD bands in the 250–300 nm region, indicating the presence of asymmetrically situated aromatic residues. On heating from 0 to 60°C, a sigmoidal conformational transition is seen to occur when monitored by means of the 225 nm CD band. Addition of 6 M guanidine hydrochloride produces a CD spectrum typical of denatured proteins. On treatment with SDS, the spectrum of the native protein changes over to that of a protein having α-helical segments. The SDS-treated protein was found to be devoid of antifreeze activity. Taken together, the CD data point to the presence of well-defined secondary and tertiary structures in the ocean pout proteins which are quite different from those obtained in other antifreeze proteins.

Circular dichroism of type 13 β-turn in linear tripeptides containing L-proline and D-alanine

Abstract The linear tripeptides tBoc-L-Prolyl-D-alanyl-L-leucine and tBoc-L-prolyl-D-alanyl-L-valine have been shown, from circular dichroism (CD) and infrared spectral data, to take up the 4 → 1 hydrogen bonded β-turn conformation in organic solvents. The CD spectra of these tripeptides in trifluoroethanol exhibit a positive n → π★ band around 220 nm contrary to the usual negative band observed for the type II β-turn. The observed CD spectra of the tripeptides provide the first examples of those predicted theoretically by Woody for peptides containing L,D sequences and adopting the Venkatachalam type 13 β-turn. This conformation is seen to revert to the type II β-turn when the N-terminal protecting group is acetyl or when the C-terminal residue is glycine. These data are shown to have a direct bearing on the interpretation of the CD spectra of globular proteins.

Role of calcium in stabilizing the structure of hyalin, a major protein component of the sea urchin extraembryonic hyaline layer.

The interactions of NaCl and CaCl2 with the sea urchin embryo coat protein hyalin were investigated. Endogenous protein tryptophan fluorescence was enhanced by almost 45% in the presence of 200mM NaCl while 1mM CaCl2 reversed this effect and brought the intensity of fluorescence back close to that of the native protein. Half-maximal concentrations of 53 and 0.32mM were determined for NaCl and Ca+2, respectively. Hyalin conformation, as measured by circular dichroic spectroscopy, was altered by NaCl and CaCl2 in a fashion parallel to the effects of these salts on tryptophan fluorescence. Sodium chloride disrupted hyalin secondary structure while CaCl2 affected the return of hyalin to its native conformation. The interactions of NaCl and CaCl2 with hyalin were not modulated by MgCl2. These results suggest a role for CaCl2 in stabilizing hyalin against the disruptive effects of the high concentration of NaCl present in sea water.