The melting behavior of the homopolymers poly[A5-(3-hydroxypropyl)-L-glutamine] and poly[(V6-(4-hydroxybutyl)-L-glutamine], and copolymers of these two amino acids, was determined in water. The homopolymer data were treated by the Zimm-Bragg theory and the copolymer data were analyzed with the lowest order approximation of the theory discussed in the previous paper. Within the experimental error, it was not possible to detect any temperature dependence of the parameter or of the parameters AH and AS for the homopolymers. Using these temperature-independent parameters for one of the homopolymers, it was possible to compute those for the other homopolymer by applying the host-guest technique (and associated theory) to the copolymer data. Good agreement was obtained between the parameters computed directly from homopolymer data and those obtained by the host-guest technique, thus establishing the validity of the latter method for future use in obtaining the helix-coil stability constants for other amino acids.

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly a-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The a-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.To avoid freezing, many species ofmarine fishes inhabiting the Newfoundland and Labrador coastal waters produce antifreeze polypeptides (AFP) in the winter months [I -31. These species include the winter flounder [4-61, shorthorn sculpin [7], sea raven [8] and ocean pout [9]. Isolation and characterization of these AFP have indicated three distinct types of polypeptides based on the differences in amino acid composition and secondary structure [4-91. The AFP from flounder and sculpin comprise one such class.The AFP from the winter flounder, Pseudopleuronectus americunus can be fractionated into a t least seven active components by reverse-phase high-performance liquid chromatography (HPLC) [6]. The two major components, each with a relative molecular mass of about 3300, are closely related. Both consist of eight or nine different amino acids of which alanine accounts for 60% of the residues [4-61. The amino acid sequence of these two components have been elucidated [lo -121. The 37-residue-long polypeptide chain in each component has been found to contain three repeating sequences of 11 amino acid residues, Thr-(X)2-Y-(X)7-, where X represents a non-polar amino acid (mainly alanine) and Y is a polar amino acid [lo-121. The AFP from the shorthorn sculpin, Myoxocephulus scorpius, are similar to those of the winter flounder with respect to the abundance of alanine in their amino acid compositions and their high cr-helical contents [7, 131. However, they contain 12 different amino acids, and have slightly larger molecular masses withCorrespondence to

In 1979 it was proposed that prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1. 14.11.2) recognizes the 3-turn conformation in nascent procollagen chains and that the hydroxylation process involves a conformational change resulting in "straightening" of the (turn segments into the linear triple-helical conformation of native collagen. We present experimental data that verify both these postulates. The following peptides were synthesized and studied for their conformation and interaction with prolyl hydroxylase: tBoc-Pro-GlyAla-OH, tBoc-Pro-Gly-Val-OH, tBoc-Gly-Val-Pro-Gly-Val-OH, and tBoc-Pro-DAla-Ala-OH. Spectral data showed that these peptides preferred a (-turn conformation. All of them acted as inhibitors of the enzyme; the pentapeptide also acted as a substrate. To mimic the biosynthetic event, a collagen model polypeptide, (Pro-Pro-Gly)10, was incubated at 37C with purified prolyl hydroxylase and the necessary cosubstrates and cofactors at pH 7.8. A progressive change from the initially nonhelical to the triplehelical conformation, as monitored by CD spectra and gel filtration, occurred during the course of proline hydroxylation. In addition to leading to increased thermal stability of the triple-helical conformation in (Pro-Pro-Gly)10 and (Pro-Pro-Gly)5, the enzymatic incorporation of the hydroxyproline residues was found to enable these polypeptides to fold into this conformation faster than the unhydroxylated counterparts. These conformational implications ofproline hydroxylation in collagen may also be of use in the study of the complement subcomponent Clq and of acetylcholine esterase which contain collagen-like regions in them.The post-translational hydroxylation of selected proline residues by prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) is a crucial event in the biosynthesis of collagen. The resulting hydroxyproline residues are essential for the stability of the triple-helical conformation of collagen at body temperature (1). In regard to the conformational aspects of the enzymatic hydroxylation process, two questions needed to be answered: (a) What is the preferred conformation of the peptide substrate? and (b) What is the conformational consequence of proline hydroxylation? Based on experimental data and theoretical considerations, it was proposed (2) that prolyl hydroxylase recognizes the (3-turn conformation (3) formed at the -Pro-Gly-segments in the nascent procollagen chains and that the hydroxylation process results in "straightening" of these segments into the rigid conformation necessary for the subsequent association of these chains in the triple-helical conformation of native collagen.In order to test these postulates in a direct manner, we undertook (a) the synthesis of simple peptides which were expected to prefer the (3-turn conformation to study their interactions with prolyl hydroxylase and (b) MATERIALS AND METHODS Enzyme. Prolyl hydroxylase from 13-day chicken embryos was purified to hom...

Obtained by successive approximations in the refinement of the homopolymer and copolymer data. for homopolymers alone; this may tend to overweight the copolymer data so that one loses sight of the behavior of the homopolymers. However, we find that the PHBG parameters obtained in this manner differ little from those obtained using only the homopolymer data; hence, the PHBG homopolymer parameters in Table II appear to be good choices when PHBG is used as the host. The PHPG parameters obtained from the refinement are somewhat different from those obtained using only the homopolymer data. This is not unexpected since, as stated above, only the high-temperature ends of the melting curves are obtainable when the solvent is water.to be correct and a best fit was obtained to the experimental data for both PHBG and the copolymers. After several such cycles of refinement, the data converged to the results given in Table V for the homopolymers. It should be emphasized that the data of Table V are the result of curve fitting of data for copolymers and homopolymers rather than

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