Earlier studies have indicated that the N- and C-terminal regions of glucagon are functionally and structurally different. We have sought to understand this distinction in terms of the interaction of glucagon and its N- and C-terminal fragments with Ca2+, Mg2+, and Zn2+ in a nonpolar milieu. CD spectral data, in 98% (v/v) trifluoroethanol in water, reveal two binding sites for Ca2+ and Mg2+ and one site for Zn2+ in the intact hormone as well as in the C-terminal 19-29 fragment. The 1-6 fragment did not bind Zn2+ and formed a 2:1 peptide-Ca2+ or -Mg2+ complex. With glucagon and the 19-29 fragment, cation binding caused changes in the peptide's helix content. Fluorescence spectral changes involving Trp-25 in the 19-29 fragment and Trp-25 and Tyr-10 and/or Tyr-13 in glucagon were seen on Ca2+ binding to one of the two sites, while Zn2+ binding produced no change in fluorescence. The spectral data suggest that Ca2+ and Zn2+ binding sites (with Kd in the micromolar range in 98% trifluoroethanol) are distinct and are contained in the C-terminal domain of glucagon. Glucagon and the 19-29 fragment, but not the 1-6 fragment, caused an influx of Ca2+ (as monitored by spectral changes in arsenazo III) in unilamellar vesicles made of dimyristoyllecithin. Leakage of vesicle contents induced by the 19-29 fragment was minimal but was significant (approximately 10%) in the case of glucagon. The transport data suggest an interaction of the C-terminal domain of glucagon with Ca2+ at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.

Extracellular Ca2+ is necessary for the action of gonadotropin‐releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca2+ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca2+ under membrane‐mimetic conditions. The peptides used, in increasing order of their reported gonadotropin‐releasing activities, were: des‐amide GnRH (or GnRH‐OH); [Ala6]GnRH; [d‐Ala6]GnRH; des‐Gly10[d‐Ala6,Pro9‐NHEt]GnRH and, des‐Gly10[d‐Trp6,Pro9‐NHEt]GnRH. Changes in the far‐UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca2+‐binding isotherms. The data show that GnRH and its active analogs contain two Ca2+ binding sites, whereas the inactive analogs have only one. The extent of Ca2+ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des‐Gly10[d‐Trp6,Pro9‐NHEt]GnRH exhibited the ability to transport Ca2+ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca2+. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca2+ in the lipid milieu.