No abstract

Helix-Coil Stability Constants for Amino Acids in Water 177 Comparison of the Thermal Reaction Rate in Bulk and in Dilute Solution. It is at first sight surprising that the thermal isomerization of side chains of polymers in the rubbery state should proceed as easily as in dilute solution, since changes in the geometry of such bulky groups must require extensive rearrangements in the conformation of neighboring chain molecules. However, the following argument seems to account for the observed behavior. The conversion from a cis to a trans azo group should not be thought of as taking place in a single step, but as a result of many small oscillations of the _N-N-C bond angle.22 Although the increase of this angle should be slowed down by the requirement that neighboring chains assume a conformation allowing such a change, the reverse process should bé impeded to the same extent.

Hsp47, a 47 kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6-7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10 min and levels off by approx. 60 min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2-7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in alpha-helix and an increase in beta-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions.

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca 2؉ and, consequently, adopts a conformation that is essential for its ice-binding activity.Many marine teleost fishes are protected from freezing in icy sea water by antifreeze proteins (AFPs) 1 or glycoproteins (AFGPs). These proteins lower the freezing points of solutions in a noncolligative manner by inhibiting the growth of ice crystals (Ananthanarayanan, 1989;Davies and Hew, 1990). Four distinct types of antifreezes have been found in fishes, and each has a narrow phyletic distribution. The AFGPs are found in the cods and in the nototheniids (see Davies and Hew (1990) and references therein), and they consist of a series of repeated tripeptide units (Ala-Ala-Thr) with an O-linked disaccharide linked to each Thr residue. The type I AFPs, found in sculpins and in righteye flounders, are Ala-rich amphipathic ␣-helices (Davies and Hew, 1990). The type II AFPs are larger proteins with a folded structure and are found in smelt (Osmerus mordax), herring (Clupea harengus harengus), and sea raven (Hemitripterus americanus) (Ewart et al., 1992;Ng and Hew, 1992;Ewart and Fletcher, 1993). Type III AFP is a protein with a ␤ sandwich structure and appears limited to eel pouts (Sön-nichsen et al., 1993).The type II AFPs from sea raven, smelt, and herring share a high protein sequence identity. All three proteins are homologous to the carbohydrate recognition domains (CRDs) of Ca 2ϩ -dependent (C-type) lectins and correspond to the group VII C-type lectin family (Drickamer and Taylor, 1993). Multiple sequence alignment of the sea raven AFP with C-type CRDs and modeling of the AFP using coordinates from the CRD of rat mannose-binding protein (Weis et al., 1991a) has confirmed the structural similarity of the type II AFP to the C-type CRD fold (Sönnichsen et al., 1995). However, in contrast to the lectins, the AFPs do not bind to carbohydrates .Two Ca 2ϩ -binding sites were identified in the crystal structure of a C-type CRD from rat mannose-binding protein (MBP), and a single site was identified in the CRD of E-selectin (Weis et al., 1991a;Graves et al., 1994). The Ca 2ϩ -binding site that is shared by the selectin and the MBP was shown to be the carbohydrate-binding surface in a crystal structure of an oligosaccharide-MBP complex (Weis et al., 1992). Like the C-type lectins, the herring and smelt AFPs require Ca 2ϩ for their activity (Ewart et al., 1992;Ewart and Fletcher, 1993). In order to understand the role of Ca 2ϩ in the structural integrity and activity of these AFPs and the structural alterations underlying the evolution from carbohydrate to ice binding, we examined the role of Ca 2ϩ in the conformation and the antifreeze activity of the herring AFP (hAFP). Our findings show that hAFP contains a single Ca . EXPERIMENTAL PROCEDURESMaterial...