The Glycine1 Residue in Cyclic Lactam Analogues of Galanin(1-16)-NH2 Is Important for Stabilizing an N-Terminal Helix
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- 作者：Katharine A. Carpenter ; Ralf Schmidt ; Shi Yi Yue ; Lejla Hodzic ; Chantevy Pou ; Kemal Payza ; Claude Godbout ; William Brown ; and Edward Roberts
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：November 16, 1999
- 年：1999
- 卷：38
- 期：46
- 页码：15295 - 15304
- 全文大小：135K
- 年卷期：v.38,no.46(November 16, 1999)
- ISSN：1520-4995
文摘
The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions aremediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associatedwith the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out onfull-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide'sN-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attemptto stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformationalproperties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescencespectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied.Both c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in themicelle solution. On the basis of the analyses of their respective 
H chemical shifts and NOE patterns,this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9in c[D4,K8]Gal(1-16)-NH2 was determined to be 10.8 ± 3 Å from fluorescence resonance energy transfermeasurements. This interchromophore spacing was found to be more consistent with a helical structurethan an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potencyat the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained forthe Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within theN-terminal sequence. Decreasing the ring structure size in c[D4,K8]Gal(1-16)-NH2 by replacing Lys8with an ornithine residue or by changing the position of the single lysine residue from eight to seven wasaccompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It isconcluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ringstructure size and the presence of an N-terminal glycine residue are important for stablilizing an N-terminalhelix in these compounds. However, although an N-terminal helix constitutes a predominant portion ofthe conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2, thesepeptides nevertheless are able to adopt other conformations in solution. Consequently, the correlationbetween the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1receptor may be considered as a working hypothesis.
H chemical shifts and NOE patterns,this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9in c[D4,K8]Gal(1-16)-NH2 was determined to be 10.8 ± 3 Å from fluorescence resonance energy transfermeasurements. This interchromophore spacing was found to be more consistent with a helical structurethan an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potencyat the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained forthe Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within theN-terminal sequence. Decreasing the ring structure size in c[D4,K8]Gal(1-16)-NH2 by replacing Lys8with an ornithine residue or by changing the position of the single lysine residue from eight to seven wasaccompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It isconcluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ringstructure size and the presence of an N-terminal glycine residue are important for stablilizing an N-terminalhelix in these compounds. However, although an N-terminal helix constitutes a predominant portion ofthe conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2, thesepeptides nevertheless are able to adopt other conformations in solution. Consequently, the correlationbetween the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1receptor may be considered as a working hypothesis.