Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance

doi:10.1016/S0022-2836(83)80143-0

Journal of Molecular Biology

Volume 169, Issue 4, 5 October 1983, Pages 921-948

https://doi.org/10.1016/S0022-2836(83)80143-0 Get rights and content

A determination of the spatial structure of the polypeptide hormone glucagon bound to perdeuterated dodecylphosphocholine micelles is described. A map of distance constraintsbetween individually assigned hydrogen atoms of the polypeptide chain was obtained from two-dimensional nuclear Overhauser enhancement spectroscopy. These data were used as the input for a distance geometry algorithm for computing conformations that would be compatible with the experiments. In the region from residues 5 to 29 the mobility of the polypeptide backbone and most of the amino acid side-chains was found to be essentially restricted to the overall rotational tumbling of the micelles. The secodary structure in this region includes three turns of irregular α-helix in the segment of residues 17 to 29 near the C terminus, a stretch of extended polypeptide chain from residues 14 to 17, an α-helix-like turn formed by the residues 10 to 14 and another extended region from residues 5 to 10. In the N-terminal tetrapeptide H-His-Ser-Gln-Gly- the two terminal residues are highly mobile, indicating that they extend into the aqueous phase, and the mobility of the residues Gln3 and Gly4 appears to be only partially restricted by the binding to the micelle. The absence of long range nuclear Overhauser effects between the peptide segments 5–9 and 11–29, and between 5–16 and 19–29 shows that the polypeptide chain does not fold back on itself and hence that micelle-bound glucagon does not adopt a globular tertiary structure. Previously it was shown that the polypeptide backbone of glucagon is located close to and runs roughly parallel to the micelle surface. Combination of these observations suggests that the overall spatial arrangement of the glucagon polypeptide chain in a lipid-water interphase is largely determined by the topology of the lipid support, in the present case the curvature of the dodecylphosphocholine micelles. The tertiary structure is further characterized by the formation of two hydrophobic patches by the side-chains of Phe6, Tyr10 and Leu14, and the side-chains of Ala19, Phe22, Val23, Trp25 and Leu26, respectively.

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Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic. It is important to reveal the glucagon-membrane interaction to understand activity and cytotoxicity of glucagon and glucagon oligomers. In this review, first glucagon-membrane interactions are described as morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in acidic and neutral conditions as compared to the case of melittin. Second, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes. Third, kinetic analysis of glucagon fibril formation was performed using a two-step autocatalytic reaction mechanism, as investigated in the case of human calcitonin. The first step is a nuclear formation, and the second step is an autocatalytic fibril elongation. Forth, fibril formation of glucagon inside glucagon-DMPC bilayers in neutral solution under near physiological condition is described.
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The four publications describe the foundations for obtaining sequence-specific resonance assignments and describe complete sets of resonance assignments for BPTI and for glucagon bound to DPC micelles [65–68]. Having thus solved the NMR assignment problem, a structure of micelle-bound glucagon was completed [69] (see below), but it took two more years for the development of improved algorithms and their implementation for distance geometry calculations [44,45,47] that could yield the first complete structure determination of a globular protein, bull seminal protease inhibitor (BUSI) [1]. During the last phase of the methods development for protein structure determination by NMR in solution, from 1981 to 1985, we worked with glucagon bound to DPC micelles, BPTI, BUSI and metallothionein (MT).
This paper presents my recollections on the development of protein structure determination by NMR in solution from 1968 to 1992. The key to success was to identify NMR-accessible parameters that unambiguously determine the spatial arrangement of polypeptide chains. Inspired by work with cyclopeptides, model considerations showed that enforcing short non-bonding interatomic distances imposes «ring closure conditions» on polypeptide chains. Given that distances are scalar parameters, this indicated an avenue for studies of proteins in solution, i.e., under the regime of stochastic rotational and translational motions at frequencies in the nanosecond range (Brownian motion), where sharp pictures could not be obtained by photography-related methods. Later-on, we used distance geometry calculations with sets of inter-atomic distances derived from protein crystal structures to confirm that measurements of short proton—proton distances could provide atomic-resolution structures of globular proteins. During the years 1976–1984 the following four lines of research then led to protein structure determination by NMR in solution. First, the development of NMR experiments enabling the use of the nuclear Overhauser effect (NOE) for measurements of interatomic distances between pairs of hydrogen atoms in proteins. Second, obtaining sequence-specific resonance assignment solved the “phase problem” for protein structure determination by NMR. Third, generating and programming novel distance geometry algorithms enabled the calculation of atomic-resolution protein structures from limited sets of distance constraints measured by NMR. Fourth, the introduction of two-dimensional NMR provided greatly improved spectral resolution of the complex spectra of proteins as well as efficient delineation of scalar and dipole–dipole ¹H–¹H connectivities, thus making protein structure determination in solution viable and attractive.
31P and 13C solid-state NMR analysis of morphological changes of phospholipid bilayers containing glucagon during fibril formation of glucagon under neutral condition
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Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing ³¹P NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using ¹³C solid-state NMR spectroscopy. α-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to β-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k₁), and the second step is an autocatalytic heterogeneous fibrillation process (k₂).
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Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α−cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. To understand mechanism of fibril formation, we investigated the structure and kinetics of glucagon fibril formation using ¹³C solid-state NMR spectroscopy. In aqueous acetic acid solution at pH 3.3, distorted α−helical structure appeared around Gly4, Leu14, Ala19 and Leu26 in the monomeric form. In contrast, Gly4 and Ala19 were involved in β-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k₁), and the second step is an autocatalytic heterogeneous fibrillation process (k₂). The rate constants k₁ and k₂ were separately determined in the acetic acid solution. Fibril formation was further investigated in the presence of lipid bilayers to mimic the physiological condition. We used bicelles which form discoidal nano-particles as the bilayer system and observed that the N-terminal α-helix did not change to β-sheet when fibrils formed in the presence of bicelles. Rate constant k₁ became faster and k₂ became slower in the presence of bicelles compared to the case in the absence of bicelles. Our findings reveal that the structure and kinetics of fibril formation by glucagon are altered in the presence of lipid bilayers.
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In addition, the C-terminal helix is much shorter than desired. Long range contacts probed by nuclear Overhauser enhancement (NOE) effects (Braun et al., 1983) were shown in Fig. 3. The long range NOEs between the different β-strands are present and consistent with the arrangement of the strands within the sheet: β2 and β3 aligned in parallel, β4 and β5 form a hairpin, and the two parts are combined with β2 and β4 aligned in an anti-parallel way.
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^*: Financial support was obtained from the Schweizerischer Nationalfonds (project 3.528.79) and through a special grant of the ETH Zürich.

^†: Present address: Dept of Physics, Kyushu University 33, Fukuoka 812, Japan.

^‡: Present address: Spectrospin AG, Industriestrasse 26, CH-8117 Fällanden, Switzerland.

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Cited by (302)

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<sup>31</sup>P and <sup>13</sup>C solid-state NMR analysis of morphological changes of phospholipid bilayers containing glucagon during fibril formation of glucagon under neutral condition

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Proteins of well-defined structures can be designed without backbone readjustment by a statistical model

Journal of Molecular Biology

Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance*

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Conformation of glucagon in a lipid-water interphase by ¹H nuclear magnetic resonance*