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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biomol NMR Assign. Author manuscript; available in PMC 2017 April 1.
Published in final edited form as:
PMCID: PMC4789158

Backbone and side chain chemical shift assignments of apolipophorin III from Galleria mellonella


Apolipophorin III, a 163 residue monomeric protein from the greater wax moth Galleria mellonella (abbreviated as apoLp-IIIGM), has roles in upregulating expression of antimicrobial proteins as well as binding and deforming bacterial membranes. Due to its similarity to vertebrate apolipoproteins there is interest in performing atomic resolution analysis of apoLp-IIIGM as part of an effort to better understand its mechanism of action in innate immunity. In the first step towards structural characterization of apoLp-IIIGM, 99% of backbone and 88% of side chain 1H, 13C and 15N chemical shifts were assigned. TALOS+ analysis of the backbone resonances has predicted that the protein is composed of five long helices, which is consistent with the reported structures of apolipophorins from other insect species. The next stage in the characterization of apoLp-III from G. mellonella will be to utilize these resonance assignments in solving the solution structure of this protein.

Keywords: apolipophorin III, exchangeable apolipoprotein, apoLp-III, Galleria mellonella, NMR, chemical shift assignment

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that reversibly associates with diacylglycerol-enriched lipoproteins in insect hemolymph. The protein is relatively small (~18 kDa) and monomeric in the lipid-free form, although up to 16 apoLp-III can cluster on the lipoprotein surface during insect flight when diacylglycerol transport is greatly stimulated. Due to structural similarities with vertebrate apolipoproteins apoLp-III is used as a prototype to investigate the structure and function of exchangeable apolipoproteins (Narayanaswami et al. 2010). Like many apolipoproteins, apoLp-III is able to solubilize phospholipid vesicles into discoidal complexes, and this is mediated by the presence of amphipathic α-helices, a common structural feature of apolipoproteins.

X-ray and NMR solution structures have been solved for apoLp-III from the African migratory locust Locusta migratoria and the tobacco hornworm Manduca sexta (Breiter et al. 1991; Wang et al. 2002; Fan et al. 2003). These species are unrelated and their apoLp-III proteins share less than 12% amino acid sequence identity. However, they display a similar globular fold of a bundle of five amphipathic α-helices organized in an up-and-down topology. Both proteins have been extensively studied for their role in lipid transport processes in vivo and to improve our understanding of apolipoprotein structural changes upon interaction with lipid surfaces (Weers et al. 2006; Van der Horst et al. 2010). A third apoLp-III, of interest here, is primarily studied for its function in innate immunity (Zdybicka-Barabas et al. 2013a). This apoLp-III, from the greater wax moth Galleria mellonella (and abbreviated as apoLp-IIIGM), upregulates expression of antimicrobial proteins, associates with lipopolysaccharides, and binds and deforms bacterial membranes (Gotz et al. 1997; Weise et al. 1998; Oztug et al. 2012; Zdybicka-Barabas et al. 2013b). G. mellonella and M. sexta are from the Sphingidae family, and apoLp-III from these species have a high degree of sequence similarity (Weise et al. 1998).

Due to the increasing interest in the function of exchangeable apolipoproteins in innate immunity, it is critical to have a detailed knowledge of the three-dimensional structure of apoLp-IIIGM. A structure-guided approach would be extremely beneficial in order to improve our understanding of its novel function. As the first step in the structural characterization of this protein, backbone and side chain 15N, 1H and 13C chemical shift assignments were carried out.

Methods and Experiments

Protein expression and purification

All isotopes were obtained from Cambridge Isotope Laboratories.

Recombinant apoLp-IIIGM was expressed at 37 °C in an established bacterial expression system using BL21 cells in M9 minimal media (47.75 mM sodium phosphate, 22 mM potassium phosphate, 8.65 mM sodium chloride, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.25% U-13C-glucose-C6 (m/v), 18.7 mM 15NH4Cl, pH 7.2) (Niere et al. 1999). When the OD600 reached 0.6, over-expression was induced with the addition of IPTG to a final concentration of 1 mM and the cells were then grown at 37°C for 4 additional hours before harvesting. Cells were removed by centrifugation at 7,000×g for 15 min, and the supernatant was concentrated with a regenerated cellulose membrane (10 kDa) in a stirred ultrafiltration cell (Millipore). ApoLp-IIIGM was purified by size-exclusion chromatography using Sephadex G-75 resin (GE Healthcare). Further purification was achieved by RP-HPLC (Ultimate 3000) HPLC system, Thermo Scientific) using a Zorbax 300SB-C8 5µm 9.4×250 mm column (Agilent Technologies). A linear gradient of water and acetonitrile in 0.05% TFA (v/v) was used for protein elution. To remove solvent, the apoLp-IIIGM fraction was freeze-dried; the protein was then dissolved in 6 M guanidine-HCl and dialyzed against 3 L of 200 mM potassium phosphate buffer, pH 6.1 at 4°C with three additional buffer changes over 48 hours.

The final NMR sample contained 1.2 mM 13C/15N apoLp-IIIGM in 200 mM potassium phosphate, pH 6.1, 2 mM NaN3, 0.2 mM DSS, 10% D2O.

Assignment experiments

All experiments were recorded at 25 °C on a 600 MHz Agilent DD2 spectrometer with a room-temperature probe. Standard assignment experiments included 2D 15N HSQC, 13C aliphatic HSQC, 13C aromatic HSQC, Hb(CbCgCd)Hd, and Hb(CbCgCdCe)He spectra, as well as 3D HNCaCb, CbCa(CO)NH, HNHA, HNCO, HCC-TOCSY-NH, CCC-TOCSY-NH, HCCH-TOCSY, aromatic HCCH-TOCSY, 15N NOESY-HSQC, 13C NOESY-HSQC and aromatic NOESY experiments. The 13C and aromatic NOESY experiments utilized 100 ms mixing times and the 15N NOESY experiment utilized a 150 ms mixing time. There were challenges in assignment due to missing resonances and ambiguity due to multiple similar sequence segments. As a result, a number of spectra were recorded in addition to these standard experiments. These included HNHb (Archer et al. 1991), HbHa(CO)NH (which correlates Hβ and Hα at (i-1) with 15N and 1HN at (i))(Grzesiek et al. 1993), HNN (which correlates 15N and 1HN at (i) with 15N and 1HN at (i+1) and 15N and 1HN at (i-1))(Panchal et al. 2001), and TOCSY-HSQC (providing intraresidue correlation of aliphatic 1H with 15N and 1HN)(Zhang et al. 1994).

Data were processed using NMRPipe v.8.2 (Delaglio et al. 1995) and analyzed with NMRViewJ v.9.0.0-b114 (Johnson 2004; Johnson 2013).

Assignments and Data Deposition

Backbone assignments for apoLp-IIIGM are 99% complete (not counting the amide nitrogens from the four Pro residues or from the N-terminus). This average is comprised of 100% amide nitrogen and hydrogen, 98% Hα, 100% Cα and 99% carbonyl carbon atoms assigned. An annotated 1H-15N HSQC is provided (Fig. 1).

Fig. 1
1H-15N HSQC spectrum of 1.2 mM 13C/15N apoLp-IIIGM in 200 mM potassium phosphate buffer, pH 6.1, recorded at 25 °C on a 600 MHz NMR spectrometer. Each peak assignment is labeled with a one-letter code and residue number. Side chain NH2 groups ...

Assignments of side chain resonances (including Cβ and Hβ), at 88%, are somewhat less complete than those of the backbone. The following were not included in the calculation of the total number of side chain atoms: labile protons and their associated heteroatoms (such as amine groups on lysines or hydroxyl groups on serines) with the exception of NH2 groups on Asn and Gln, methyl groups at the end of Met side chains, carbons that have no attached hydrogens, and groups with degenerate assignments. Many of the unassigned resonances are a result of missing lysine side chain resonances: only 65% of the 17 lysine side chain atoms were successfully assigned. If lysines are excluded from the calculation, side chain assignment of apoLp-IIIGM is 93% complete.

Although the RunAbout semi-automated chemical shift assignment tool in NMRViewJ (Johnson 2004) was employed initially, it quickly became apparent that the majority of the assignment of apoLp-IIIGM would have to be performed manually. This was due to significant resonance overlap in the center of the 1H-15N HSQC spectrum (Fig. 1), weak or missing peaks in various 3D experiments, as well as similar or repeated segments of the sequence which required in-depth interpretation and additional experiments in order to determine the correct assignments (Fig. 2a). It is notable that apoLp-IIIGM contains 17 lysine, 17 glutamine and 24 alanine residues; these three amino acids therefore represent 36% of the overall sequence (compared to 18% on average for all proteins in the July 2015 UniProt database (UniProt 2015).

Fig. 2
a) Sequence of apoLp-IIIGM; b) TALOS+ prediction of protein secondary structure, utilizing 1HN, 15N, 1Hα, 13Cα and 13Cβ chemical shifts (Shen et al. 2009).

TALOS+ analysis (Shen et al. 2009) of the backbone chemical shifts (1HN, 15N, 1Hα, 13Cα and 13Cβ) predicts that apoLp-III from Galleria mellonella contains 5 helices (Fig. 2b), which is consistent with the solved structures of apolipophorins from other species. These chemical shift assignments constitute the first step in the structural characterization of apoLp-IIIGM. Studies as described in this report (and the subsequent solution structure which is forthcoming) will provide critical insight into the mechanism by which G. mellonella apoLp-III contributes to stimulating immune processes, and may shed light onto parallel activities of apolipoproteins in humans (Oztug et al. 2012).

Backbone and side chain assignments of apoLp-IIIGM at pH 6.1 have been deposited online at the BMRB (accession number 26638).


Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (award number SC3GM089564), and by a Major Research Instrumentation grant for a 600 MHz NMR spectrometer provided by the National Science Foundation (award number CHE-1040134).


Conflict of Interest

The authors declare that they have no conflict of interest in the publication of this manuscript.

Contributor Information

Karin A. Crowhurst, Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St., Northridge CA 91330-8262, ude.nusc@tsruhworc.nirak, phone: 818-677-4288, fax: 818-677-4288.

James V.C. Horn, Department of Chemistry and Biochemistry, California State University Long Beach, 1250 Bellflower Blvd., Long Beach CA 90840-9507.

Paul M.M. Weers, Department of Chemistry and Biochemistry, California State University Long Beach, 1250 Bellflower Blvd., Long Beach CA 90840-9507.


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