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SNAREs pp 115-144 | Cite as

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering

  • Andrew E. Whitten
  • Russell J. Jarrott
  • Shu-Hong Hu
  • Anthony P. Duff
  • Gordon J. King
  • Jennifer L. Martin
  • Michelle P. ChristieEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.

Key words

Small-angle X-ray scattering Small-angle neutron scattering Neutron contrast variation Munc18:syntaxin Protein complexes Cross-linking 

1 Introduction

Vesicle fusion is driven by the formation of a complex between Soluble N-ethylmaleimide -sensitive factor Attachment protein REceptor (SNARE ) proteins on a vesicle and cognate SNARE partners on a target membrane [1, 2]. Neurotransmission, for example, is driven by the association between the SNARE proteins syntaxin 1 (Sx1), SNAP-25 at the synapse, and its cognate SNARE partner VAMP2 present on synaptic vesicles. Similarly, syntaxin4 (Sx4) and SNAP-23 at the plasma membrane interact with VAMP2 on vesicles carrying the sugar transporter GLUT-4 in muscle and adipose tissue. Of particular interest to this work is the SNARE protein syntaxin, which is formed from a short ten-residue N-terminal sequence (N-peptide), a three-helical Habc domain, a SNARE domain (that interacts with partner SNARE proteins), and a C-terminal transmembrane domain . Syntaxin is thought to exist in at least two different conformations—an open fusion competent state where the SNARE domain is able to interact with cognate SNAREs, and a closed state in which the Habc domain binds to the SNARE domain preventing it from forming a SNARE complex.

The Sec/Munc (SM) proteins regulate fusion events through their interactions with SNARE proteins [3, 4]. Although the SM proteins are essential components of the fusion machinery, their precise role—positive or negative—is widely debated. The opposing functions assigned to SM proteins are partly due to the different binding modes identified from structural studies of SM:Sx complexes. X-ray crystallography studies show that neuronal Munc18a binds Sx1 in a closed conformation that is unable to interact with partner SNARE proteins [5, 6, 7, 8]. In a second more conserved binding mode SM proteins interact with the N-peptide of syntaxins [9, 10, 11]. Other work has shown that SM proteins can also interact with SNARE complexes [12, 13, 14]. For Sx1 and Sx4, the SM:SNARE complex interaction requires the Sx N-peptide interaction [13, 15] and an open conformation of the Sx [12, 14, 15, 16, 17, 18]. However, no high-resolution structures of SM proteins bound to Sx in an open conformation are available. We describe here a protocol for small-angle X-ray and neutron experiments undertaken to study the SM protein:Sx complexes of M18a:Sx1 involved in neurotransmission and Munc18c:Sx4 involved in GLUT4 transport in response to insulin signaling. In the absence of high-resolution structures the scattering data provided insights into the disposition of Sx when in complex with SM proteins . We also present protocols for chemical cross-linking experiments and describe how this information can be combined with scattering data to model a low-resolution structure for the M18:Sx complex [19].

1.1 The Small-Angle Scattering Experiment

A brief introduction to small-angle scattering, relevant to the study of Munc18:syntaxin, is given but in-depth descriptions relating to the preparation of samples and analysis of small-angle scattering can be found elsewhere [20, 21, 22, 23, 24].

Small-angle scattering involves the measurement of scattered radiation (e.g., X-rays, neutrons, or light) after interaction with a sample. The scattered radiation gives an intensity profile, I(q), that is characteristic of the structure of the particles present in solution (where q = 4π sin θ/λ). After correcting for solvent scattering, I(q) can be expressed as

$$ I(q)=N{\left(\Delta \rho V\right)}^2P(q)S(q), $$
(1)

where N is the number of particles per unit volume, Δρ is the contrast, and V is the volume of each particle. P(q) is the form factor and encodes the average structure of the particles in reciprocal space. S(q) is the structure factor and encodes the correlation distance between particles in reciprocal space. When measurements are performed on dilute solutions at micromolar concentrations the interparticle interaction is negligible so that S(q) = 1, thus providing a direct relationship between I(q) and P(q).

1.2 Neutron Contrast Variation

Small-angle X-ray scattering experiments alone yield limited information regarding the shape and disposition of individual proteins in a multicomponent system such as the SM:Sx protein complex. In such a case, neutron contrast variation provides additional information on each component of the system.

The contrast of a particle (Δρ) can be defined as the difference between the average scattering density of the particle, 〈ρ(r)〉, and that of the medium surrounding it, ρs:

$$ \Delta \rho =\left\langle \rho \left(\mathbf{r}\right)\right\rangle -{\rho}_{\mathrm{s}}, $$
(2)
and represents the average scattering power of the particle per unit volume. Unlike X-rays that are scattered predominantly by electrons, neutrons are scattered primarily by the nucleus. Therefore, neutron contrast is dependent on the isotopic composition of the scattering particle and the surrounding medium and can be changed by isotopic labeling with deuterium of one component of a complex. The contrast of each component can be further enhanced or diminished by varying the 1H:2H ratio of the solvent, so that the contrast of a given component in the complex can be adjusted to zero—known as contrast matching . The contrast-matched component is essentially invisible to neutrons, and allows scattering data to be interpreted in terms of the visible component alone (Fig. 1). In our case we labeled Sx1 and Sx4, and used contrast matching to match out Sx scattering to observe the bound conformation of Munc18, or to match out Munc18 scattering to observe the bound conformation of the Sx.
Fig. 1

The contrast variation experiment. One component of a complex is preferentially deuterated and neutron scattering experiments are conducted in buffers with different 1H2O:2H2O ratios. This changes the contribution of each component to the overall scattering profile. At 100% 1H2O scattering is predominantly by both components. At 40% 2H2O scattering is predominantly by the deuterated component while at 100% 2H2O it is primarily by the non-deuterated component (reproduced from [40])

1.3 Information Derived from Small-Angle Scattering Experiments

Analysis of the scattering data from small-angle scattering experiments yields information on structural parameters of molecules. Analysis techniques include the Guinier plot [25], and indirect Fourier transformation of I(q) to obtain the P(r) function [26, 27], which is indicative of the shape of the molecule. The intensity at zero angle, I(0), is related to the molecular mass of the molecule [28]. The radius of gyration, Rg, is related to particle shape and can be obtained from the Guinier plot or P(r), where a small Rg is indicative of a compact structure and a large Rg is indicative of an extended structure.

In addition, it is possible to optimize low-resolution models of the structure of the complexes against scattering data by shape restoration (ab initio modeling ) or rigid body modeling [29, 30]. Ab initio modeling typically involves optimization of the distribution of “dummy residues” against the scattering data [30]. It is particularly useful for obtaining information on the shape and arrangement of protein complexes when no high-resolution structures are available. In rigid body modeling, which is the focus of this work, the orientation and position of an ensemble of known atomic structures comprising the molecule of interest are optimized against the scattering data [29]. Additional biochemical or biophysical information on the system under study can be incorporated to provide constraints or to validate models [24, 31, 32].

2 Materials

2.1 Constructs

Constructs used in the experiments are shown in Table 1. Details are provided below.
Table 1

Constructs used in the scattering experiments

Proteins

Organism

Residues

Vectors

Tags

N- or C-terminal tags

Cleavage site in tags

Expression system

Munc18a-His

Rattus norvegicus

1–594

pET28a

6×-His

C-

No

E. coli

Munc18c (insect)

Mus musculus

1–592

pAc- HLT-B

6×-His and a 53 a.a. Tag

N-

Thrombin

S. frugiperda ( Sf9 cells )

Sx11–261-His

Rattus norvegicus

1–261

pET24a

6×-His

C-

No

E. coli

ΔNSx125–261-His

Rattus norvegicus

25–261

pET24a

6×-His

C-

No

E. coli

Sx41–275-His

(C141S)

Rattus norvegicus

1–275

pET20b

6×-His

C-

No

E. coli

Rat Munc18a-His (residues 1–594, accession code: NP_037170) was cloned into pET28a to generate a C-terminal 6xHis-tag construct.

Full-length mouse Munc18c (residues 1–592, accession code: NP_035634) was engineered into the baculovirus transfer vector pAcHLTB between the Not1 and NcoI restriction sites for expression in insect cells. The vector sequence included a region encoding a 53-residue N-terminal leader sequence containing a six-residue His-tag, protein kinase A site, and thrombin cleavage site (underlined): MSPIDPMGHHHHHHGRRRASVAAGILVPRGSPGLDGIYARGIQASMAAGFGMQ [33]. The thrombin-cleaved construct is referred to as detagged Munc18c.

The cytoplasmic regions (lacking the transmembrane domain ) of all the Sxs were expressed in E. coli with an engineered C-terminal 6x-His tag [19]. PCR fragments of rat Sx1a (residues 1–261, accession code: NP_446240.2) and Sx1aΔN (residues 25–261) were generated by amplification of a synthetic Sx1a gene codon optimized for expression in E. coli (GeneArt). Sx41–275 was cloned into pET20b (accession code: NP_112387.1) and Sx11–261 was cloned into pET24a. Sx1 with an N-terminal deletion, Sx1ΔN (residues 25–261), was cloned into pET24a.

2.2 Media for the Expression of Non-deuterated Proteins

  1. 1.

    Luria–Bertani (LB) media: Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride—make up to 1 L in distilled water and autoclave.

     
  2. 2.

    LB agar: Weigh 15 g agar, add to 1 L of LB media, and autoclave. Allow agar media to cool to 55 °C before adding desired concentration of antibiotic. Mix well, but minimize bubbles, and pour into Petri dishes. Allow to set at room temperature, and then store at 4 °C.

     
  3. 3.

    20× NPS: Weigh 132 g ammonium sulfate, 272 g potassium dihydrogen phosphate, and 284 g disodium hydrogen phosphate anhydrous—make up to 1 L in distilled water and autoclave.

     
  4. 4.

    50× 5052: Weigh 250 g glycerol, 100 g α-lactose monohydrate, and 25 g glucose—make up to 1 L in distilled water and autoclave.

     
  5. 5.

    0.1 M Iron (III) chloride: Weigh 13.52 g iron (III) chloride hexahydrate—add 5 mL (1/100 volume) of concentrated HCl (37% w/v), make up to 500 mL in distilled water, and sterilize using a 0.2 μm filter. Do not autoclave.

     
  6. 6.

    1 M Calcium chloride: Weigh 14.7 g calcium chloride dihydrate—make up to 100 mL in distilled water and autoclave.

     
  7. 7.

    1 M Manganese chloride: Weigh 19.79 g manganese chloride tetrahydrate—make up to 100 mL in distilled water and autoclave.

     
  8. 8.

    1 M Zinc sulfate: Weigh 28.76 g zinc sulfate heptahydrate—make up to 100 mL in distilled water and autoclave.

     
  9. 9.

    0.2 M Cobalt chloride: Weigh 4.76 g cobalt chloride hexahydrate—make up to 100 mL in distilled water and autoclave.

     
  10. 10.

    0.1 M Copper chloride: Weigh 1.71 g copper chloride dihydrate—make up to 100 mL in distilled water and autoclave.

     
  11. 11.

    0.1 M Sodium molybdate: Weigh 2.42 g sodium molybdate dihydrate—make up to 100 mL in distilled water and autoclave.

     
  12. 12.

    0.1 M Sodium selenite: Weigh 1.73 g sodium selenite anhydrous—make up to 100 mL in distilled water and autoclave.

     
  13. 13.

    0.1 M Boric acid: Weigh 0.62 g boric acid—make up to 100 mL in distilled water and autoclave.

     
  14. 14.

    1000× Trace metals: Combine 36 mL sterile distilled water, 50 mL 0.1 M iron (III) chloride, 2 mL 1 M calcium chloride, 1 mL 1 M manganese chloride, 1 mL 1 M zinc sulfate, 1 mL 0.2 M cobalt chloride, 2 mL 0.1 M copper chloride, 1 mL 0.1 M sodium molybdate, 2 mL 0.1 M disodium selenite, and 2 mL 0.1 M boric acid. Filter solution through a 0.2 μm filter (do not autoclave), and store in the dark or wrapped in foil.

     
  15. 15.

    ZY Media: Weigh 10 g tryptone, and 5 g yeast extract—make up in 928 mL in distilled water and autoclave.

     
  16. 16.

    1 M Magnesium sulfate: Weigh 24.65 g of magnesium sulfate heptahydrate—make up to 100 mL in distilled water and autoclave.

     
  17. 17.

    Autoinduction media: Combine 50 mL 20× NPS, 20 mL 50× 5052, 1 mL 1000× trace metals, and 2 mL 1 M magnesium sulfate with 928 mL of autoclaved ZY media along with the appropriate antibiotic immediately prior to adding in the bacterial culture.

     
  18. 18.

    Sf900-II serum-free media.

     

2.3 Media and Reagents for the Expression of Deuterated Proteins

  1. 1.

    M9 minimum salt medium: Weigh 12.8 g disodium hydrogen phosphate anhydrous, 3.0 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 1.0 g ammonium chloride, and 2 g glucose. To that add 2 mL of 1 M magnesium sulfate, 1 mL of 0.1 M calcium chloride, 1 mL of 0.02 M thiamine, and 1 mL of 0.03 M iron (II) sulfate—make up in 1 L of either 1H2O, 50% 2H2O, 70% 2H2O, 90% 2H2O, or 99% 2H2O and filter sterilize using 0.2 μm filter (see Note 1 ).

     
  2. 2.

    0.02 M Thiamine: Weigh 0.67 g thiamine hydrochloride—make up to 100 mL in distilled water and sterilize using 0.2 μm syringe filter. Aliquot and store at −20 °C.

     
  3. 3.

    0.03 M Iron (II) sulfate: Weigh 0.83 g iron (II) sulfate heptahydrate—make up to 100 mL in distilled water and sterilize using 0.2 μm filter.

     
  4. 4.

    0.1 M Calcium chloride: Weigh 1.47 g calcium chloride dihydrate—make up to 100 mL in distilled water and autoclave.

     
  5. 5.

    1 M Isopropyl-β-D-thiogalactopyranoside (IPTG): Weigh 2.38 g of isopropyl-β-D-thiogalactopyranoside—make up to 10 mL in distilled water and sterilize using 0.2 μm syringe filter.

     
  6. 6.

    SOC media (Super Optimal Broth plus glucose): Weigh 20.0 g tryptone, 5.0 g yeast extract, 0.6 g sodium chloride, 0.2 g potassium chloride, 2.0 g magnesium chloride hexahydrate, 2.5 g magnesium sulfate heptahydrate, and 3.6 g glucose—make up to 1 L in distilled water and autoclave.

     
  7. 7.

    Bioreactor 1 L capacity (see Note 2 ).

     
  8. 8.

    Base solution: Weigh 2.6 g ammonium chloride, 2.5 g potassium dihydrogen phosphate, 4.2 g disodium hydrogen phosphate anhydrous, 1.9 g potassium sulfate, and 40.0 g glycerol—make up to 1 L in distilled water and filter sterilize using a 0.2 μm filter.

     
  9. 9.

    Additive A 1000×: Weigh 8.8 g trisodium citrate anhydrous, and 2.0 g iron (II) sulfate heptahydrate—make up to 100 mL in distilled water and sterilize using a 0.2 μm filter.

     
  10. 10.

    Additive B 1000×: Weigh 0.59 g manganese (II) sulfate monohydrate, 0.86 g zinc (II) sulfate heptahydrate, and 0.07 g copper (II) sulfate pentahydrate—make up to 100 mL in distilled water and sterilize using a 0.2 μm filter.

     
  11. 11.

    Additive C 1000×: Weigh 4.8 g thiamine—make up to 100 mL in distilled water, sterilize using a 0.2 μm filter, and store in the dark.

     
  12. 12.

    Additive D 1000×: Weigh 67.0 g magnesium sulfate heptahydrate—make up to 100 mL in distilled water and sterilize using a 0.2 μm filter.

     
  13. 13.

    ModCl minimal media: Add 1 mL each of additives A, B, C, and D to 1 L of base solution immediately prior to use (see Note 3 ).

     
  14. 14.

    Antifoam 204.

     

2.4 Buffers and Reagents for Protein Purification

  1. 1.

    1 M Tris: Add 121.14 g to 800 mL distilled water, pH with HCl to 7.5, and make up to 1 L. Sterilize with 0.2 μm filter.

     
  2. 2.

    1 M Magnesium chloride: Weigh 20.3 g magnesium chloride hexahydrate—make up to 100 mL in distilled water and sterilize using a 0.2 μm filter.

     
  3. 3.

    1 M Dithiothreitol (DTT): Weigh 1.54 g of DTT—make up to 10.0 mL in distilled water and filter sterilize using 0.2 μm syringe filter. Store at −20 °C.

     
  4. 4.

    Deoxyribonuclease I grade 2 from Roche 100 mg in 15 mL of DNase buffer (50% v/v glycerol, 100 mM Tris pH 7.5, 50 mM calcium chloride, 250 mM magnesium chloride, stored at −20 °C) (see Note 4 ).

     
  5. 5.

    1 M 2-Mercaptoethanol (β-ME): Add 0.7 mL β-ME to 9.3 mL distilled water and store at 4 °C (see Note 5 ).

     
  6. 6.

    Bacterial Protease Inhibitor Cocktail EDTA free.

     
  7. 7.

    1000× Ampicillin: Weigh 1.0 g ampicillin sodium salt—make up to 10 mL in 70% ethanol, and store at −20 °C.

     
  8. 8.

    1000× Kanamycin: Weigh 0.5 g kanamycin monosulfate—make up to 10 mL in distilled water, and store at −20 °C.

     
  9. 9.

    1000× Chloramphenicol: Weigh 0.34 g chloramphenicol—make up to 10 mL in 100% ethanol, and store at −20 °C.

     
  10. 10.

    SxHis Cell Lysis Buffer: 25 mM Tris pH 7.5, 150 mM sodium chloride, 10 mM imidazole, 2 mM β-ME, 0.5% v/v Triton X-100. Weigh 3.0 g Tris, 8.8 g sodium chloride, 0.7 g imidazole, and 5.0 mL Triton X-100, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M). Protease inhibitor should be added directly to the lysis solution at a rate of 100 μL per 20 g of cell pellet, and DNase at a rate of 200 μL per 20 g of cell pellet (see Notes 6 and 7 ).

     
  11. 11.

    SxHis Wash Buffer 1: 25 mM Tris pH 7.5, 500 mM sodium chloride, 10 mM imidazole, 2 mM β-ME. Weigh 3.0 g Tris, 29.2 g sodium chloride, and 0.7 g imidazole, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  12. 12.

    SxHis Wash Buffer 2: 25 mM Tris pH 7.5, 50 mM sodium chloride, 20 mM imidazole, 2 mM β-ME. Weigh 3.0 g Tris, 2.9 g sodium chloride, and 1.4 g imidazole, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  13. 13.

    SxHis Elution Buffer: 25 mM Tris pH 7.5, 50 mM sodium chloride, 300 mM imidazole, 2 mM β-ME. Weigh 3.0 g Tris, 2.9 g sodium chloride, and 20.4 g imidazole, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  14. 14.

    SxHis MonoQ A Buffer: 25 mM Tris pH 7.5, 25 mM sodium chloride, 2 mM β-ME. Weigh 3.0 g Tris, and 1.5 g sodium chloride, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Filter solution with a 0.2 μm filter and immediately prior to use add 2 mL of β-ME (1 M).

     
  15. 15.

    SxHis MonoQ B Buffer: 25 mM Tris pH 7.5, 500 mM sodium chloride, 2 mM β-ME. Weigh 3.0 g Tris, and 29.2 g sodium chloride, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.5 using HCl and make up to 1 L using distilled water. Filter solution with a 0.2 μm filter and immediately prior to use add 2 mL of β-ME (1 M).

     
  16. 16.

    Munc18a-His and His-Munc18c lysis buffer: 50 mM Phosphate pH 8.0, 300 mM sodium chloride, 10 mM imidazole, 10% v/v glycerol, 2 mM β-ME, 1% v/v Triton X-100. Weigh 6.62 g sodium hydrogen phosphate, 0.41 g sodium dihydrogen phosphate anhydrous, 17.6 g sodium chloride, 0.7 g imidazole, and 10.0 mL Triton X-100, and dissolve in ~800 mL of distilled water. Adjust pH to 8.0 with HCl or NaOH, add 100 mL glycerol, and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M). Protease inhibitor should be added directly to the lysis solution at a rate of 100 μL per 20 g of cell pellet, and DNase at a rate of 200 μL per 20 g of cell pellet.

     
  17. 17.

    Munc18a-His wash buffer 1: 50 mM Phosphate pH 8.0, 500 mM sodium chloride, 10 mM imidazole pH 8.0, 10% v/v glycerol, 2 mM β-ME. Weigh 6.62 g sodium hydrogen phosphate, 0.41 g sodium dihydrogen phosphate anhydrous, 29.2 g sodium chloride, and 0.7 g imidazole, and dissolve in ~800 mL of distilled water. Adjust pH to 8.0 with HCl or NaOH, add 100 mL glycerol, and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  18. 18.

    Munc18a-His wash buffer 2: 50 mM Phosphate pH 8.0, 500 mM sodium chloride, 20 mM imidazole pH 8.0, 10% v/v glycerol, 2 mM β-ME. Weigh 6.62 g sodium hydrogen phosphate, 0.41 g sodium dihydrogen phosphate anhydrous, 29.2 g sodium chloride, and 1.4 g imidazole, and dissolve in ~800 mL of distilled water. Adjust pH to 8.0 with HCl or NaOH and add 100 mL glycerol and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  19. 19.

    Munc18a-His elution buffer: 50 mM Phosphate pH 8.0, 300 mM sodium chloride, 300 mM imidazole, 10% v/v glycerol, 2 mM β-ME. Weigh 6.62 g sodium hydrogen phosphate, 0.41 g sodium dihydrogen phosphate anhydrous, 17.5 g sodium chloride, and 20.4 g imidazole, and dissolve in ~800 mL of distilled water. Adjust pH to 8.0 with HCl or NaOH, add 100 mL glycerol, and make up to 1 L using distilled water. Immediately prior to use add 2 mL of β-ME (1 M).

     
  20. 20.

    Munc18 size-exclusion buffer: 25 mM HEPES pH 7.0, 300 mM sodium chloride, 2 mM β-ME. Weigh 6.0 g HEPES, and 17.5 g sodium chloride, and dissolve in ~800 mL of distilled water. Adjust the pH to 7.0 using NaOH and make up to 1 L using distilled water. Filter solution with a 0.2 μm filter and immediately prior to use add 2 mL of β-ME (1 M).

     
  21. 21.

    His-Munc18c wash buffers: Same as Munc18a-His except 300 mM sodium chloride is used in wash buffers 1 and 2.

     
  22. 22.

    His-Munc18c cleavage buffer: 25 mM Tris pH 8.0, 300 mM sodium chloride, 3 mM calcium chloride, 2 mM β-ME. Weigh 0.3 g Tris and 1.75 g sodium chloride, add 300 μL 1 M calcium chloride, and make up to 100 mL with distilled water. Filter solution with a 0.2 μm filter and immediately prior to use add 2 mL of β-ME (1 M).

     
  23. 23.

    His-Munc18c elution buffer: His-Munc18c wash buffer with the addition of 200 mM imidazole.

     
  24. 24.

    Thrombin: Make up with 1 mL distilled water to 1 U/μL. Freeze and store −20 °C in 50 μL aliquots.

     
  25. 25.

    AEBSF protease inhibitor 400 mM: Weigh 500 mg 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and make up to 5 mL in distilled water. Aliquot and store at −20 °C.

     
  26. 26.

    Bio-Rad Protein Assay.

     

2.5 Components/Equipment Required for Protein Purification

  1. 1.

    Metal affinity resin.

     
  2. 2.

    Refrigerated shaking incubator.

     
  3. 3.

    Thompson Ultra Yield Flasks.

     
  4. 4.

    FPLC capable of pumping at 1 mL/min and able to do a gradient protocol.

     
  5. 5.

    Gel filtration column: We routinely use Superdex 200 16/60, Superdex 200 26/60 column from GE Healthcare, UK.

     
  6. 6.

    Anion Exchange Column (GE Healthcare, UK): MonoQ 5/50.

     
  7. 7.

    Centrifuges capable of handling volumes and speeds as stated.

     
  8. 8.

    Sonicator—capable of lysing cell suspensions as per volumes stated.

     

2.6 Reagents Required for Mass Spectrometry Experiments

  1. 1.

    1 M Tris–HCl (pH 8.5): Dissolve 12.1 g Tris and add ~90 mL of distilled water. Adjust pH to 8.5 using HCl and make up to 100 mL using distilled water.

     
  2. 2.

    Working stock formaldehyde: Add 110 μL of a 37% w/v solution of formaldehyde to 890 μL of distilled water (to be prepared fresh for each experiment).

     
  3. 3.

    1 M Dimethylamino borane: Dissolve 5.9 mg of dimethylamino borane in 100 μL of distilled water (to be prepared fresh for each experiment).

     
  4. 4.

    1 M Iodoacetamide: Dissolve 5.6 mg iodoacetamide in 30 μL distilled water.

     
  5. 5.

    10 mM DTTSP: Dissolve 6.1 mg 3,3′-dithiobis-(sulfosuccinimidylpropionate) in 1.0 mL of 50 mM acetic acid (to be prepared fresh for each experiment).

     
  6. 6.

    10 mM BS3: Dissolve 5.7 mg bis(sulfosuccinimidyl)suberate in 1.0 mL 50 mM acetic acid (to be prepared fresh for each experiment).

     
  7. 7.

    1 M HEPES: Weigh 238.3 g of HEPES and dissolve in 900 mL. Adjust to desired pH with HCl or NaOH. Sterilize with 0.2 μm filter.

     
  8. 8.

    M18:Sx complex buffer: 50 mM HEPES buffer pH 7.5 for Munc18c:Sx4 (DTSSP reaction), 25 mM HEPES buffer pH 7.0, 300 mM NaCl, 10% glycerol, and 2 mM DTT for Munc18c:Sx4 (BS3 reaction) and 25 mM HEPES (pH 8), 200 mM NaCl, 10% glycerol, and 2 mM DTT (BS3 reaction).

     
  9. 9.

    20% SDS solution from commercial source

     
  10. 10.

    1 M Tris pH 6.8: Add 12.1 g to 80 mL distilled water, adjust pH with HCl or NaOH to 6.8, and make up to 100 mL. Filter sterilize with 0.2 μm filter.

     
  11. 11.

    PAGE sample buffer 6×: 375 mM Tris–Cl (pH 6.8), 50% v/v glycerol, 9% SDS, 0.03% bromophenol blue. To make 3× stock add 18.75 mL 1 M Tris pH 6.8, 10 mL glycerol, 30 mL 20% SDS solution, and 75 mg bromophenol blue and make up to 100 mL with distilled water. Aliquot and store at −20 °C. Immediately before use, add 100 μL 1 M DTT per 1 mL sample buffer. When making up your gel sample dilute 1 in 3 with protein sample and/or water as required.

     
  12. 12.

    BS3 reaction buffer: 35 mM HEPES (pH 7), 300 mM sodium chloride, 10% glycerol, 2.0 mM DTT. Weigh 0.8 g HEPES and 1.75 g sodium chloride and dissolve in ~80 mL of distilled water. Add 10 mL glycerol, adjust pH to 7 using NaOH, and make up to 100 mL with distilled water. Add 200 μL of 1 M DTT immediately before use.

     
  13. 13.

    Protein stock solutions were stored at −80 °C at ~1.2 μM for experiments with DTSSP and ~2.0 μM for experiments with BS3. Solutions were thawed immediately before use.

     
  14. 14.

    MALDI-TOF matrix: Whole-protein MS: Weigh 10 mg of sinapinic acid, make up to 1.0 mL in 60% acetonitrile, and add 1 μL formic acid; peptide MS: weigh 5 mg of α-cyano-hydroxycinnamic acid, add 10 μL 0.1 M ammonium dihydrogen phosphate, and 10 μL formic acid. Make up to 1.0 mL in 60% acetonitrile.

     

2.7 Material and Equipment Used for Mass Spectrometry Experiments

  1. 1.

    C18 Zip tips.

     
  2. 2.

    MALDI plate (Applied Biosystems).

     
  3. 3.

    Vacuum concentrator.

     
  4. 4.

    Voyager DE mass spectrometer Agilent 1100 nanoHPLC.

     
  5. 5.

    4700 Proteomics Analyser

     
  6. 6.

    QSTAR Elite mass spectrometer.

     

2.8 Buffers Required for Small-Angle X-ray Acattering (SAXS) and Small-Angle Neutron Scattering (SANS) Experiments

  1. 1.

    2H2O SANS buffer: 25 mM HEPES pH 7.1, 150 mM sodium chloride, 1 mM TCEP (see Note 8 ). Weigh 3.0 g HEPES, 4.4 g sodium chloride, and 0.14 g tris(2-carboxyethyl)phosphine and dissolve in ~400 mL of 2H2O. Adjust the pH to 7.1 using a 5 M solution of NaOH in 2H2O and make up to 500 mL with 2H2O.

     
  2. 2.

    1H2O SANS/SAXS buffer: 25 mM HEPES pH 7.5, 150 mM sodium chloride, 1 mM TCEP. Weigh 3.0 g HEPES, 4.4 g sodium chloride, and 0.14 g tris(2-carboxyethyl)phosphine and dissolve in ~400 mL of distilled water. Adjust the pH to 7.1 using NaOH and make up to 500 mL with distilled water.

     

2.9 Equipment Required for the SAXS /SANS Experiments

  1. 1.

    Centrifugal concentrator (Amicon Ultra filters).

     
  2. 2.

    Dialysis cassettes (with a capacity in the order of 0.1–1.0 mL, 3.5 K MWCO).

     
  3. 3.

    Quartz cells with 1 mm path length (Hellma QS-120).

     
  4. 4.

    Protein stock solutions that were stored at −80 °C and thawed immediately prior to the experiments at a concentration of ~3 mg/mL.

     

3 Methods

3.1 Expression of Sx1 and Sx4 in E. coli

Protein expression by autoinduction method followed the protocols set out by Studier et al. [34].
  1. 1.

    Set up 10 mL starter cultures in LB media with ampicillin at 100 μg/mL and chloramphenicol at 34 μg/mL concentration, at 37 °C, 200 rpm overnight inoculated with ~10 μL of a glycerol stock of BL21(DE3)pLysS cells harboring the appropriate Sx plasmid (see Note 9 ).

     
  2. 2.

    Transfer 3 mL of the overnight starter culture into 1 L ZY-5052 autoinduction media at 30 °C, 220 rpm, and grow for up to 24 h.

     
  3. 3.

    Measure OD600 at 1:100 dilution blanked against LB media after 18–20 h. The OD600 should be >10 (see Note 10 ).

     
  4. 4.

    Harvest cells by centrifugation at 8000 × g for 10 min at 4 °C.

     
  5. 5.

    Freeze the harvested cells in liquid nitrogen and store at −80 °C.

     

3.2 Expression of ΔN–Sx1 in E. coli

  1. 1.

    Set up a 50 mL starter culture in LB media with kanamycin at 100 μg/mL and chloramphenicol at 34 μg/mL at 37 °C, 200 rpm, overnight inoculated with a streak from a fresh transformation of Rosetta2(DE3)pLysS with pET24a-ΔN-Sx1 (see Note 11 ).

     
  2. 2.

    Transfer 3 mL overnight starter culture into 300 mL ZYM-5052 autoinduction media (ZYM-5052 media has a lower concentration of phosphate (50 mM) compared with ZYP-5052 (100 mM) at 37 °C, shake at 220 rpm, and grow until OD600 is 0.5–1.0. Then lower the incubation temperature to 16 °C and allow cells to grow overnight. (see Note 12 ).

     
  3. 3.

    Measure OD600 using 1:100 dilution. The undiluted OD600 should be >10 after 18–20 h.

     
  4. 4.

    Harvest cells as described above in Subheading 3.1, steps 4 and 5.

     

3.3 Expression of Munc18a-His

  1. 1.

    Set up 10 mL starter cultures in LB media with kanamycin at 50 μg/mL (Munc18a-His) and chloramphenicol at 34 μg/mL at 37 °C, 200 rpm, overnight inoculated with ~10 μL of a glycerol stock of BL21(DE3)pLysS cells harboring the pET28a-Munc18a plasmid (see Note 9 ).

     
  2. 2.

    Use 2–5 mL overnight starter culture to inoculate 1 L of ZYM-5052 autoinduction media. Express proteins at 25 °C with shaking at 220 rpm for 24 h.

     
  3. 3.

    Measure OD600 using 1:100 dilution. The undiluted OD600 should be >10 after 18–20 h.

     
  4. 4.

    Harvest cells as described above in Subheading 3.1, steps 4 and 5.

     

3.4 Cloning and Expression of His-Munc18c in Insect Cells

  1. 1.

    Transform and amplify pAcHLT-B encoding full-length Munc18c from Escherichia coli strain DH5α.

     
  2. 2.

    Co-transfect Spodoptera frugiperda (Sf9) with the pAcHLT-B baculovirus transfer vector and BaculoGold DNA (PharMingen) according to the manufacturer’s instructions (Baculovirus Expression Vector System Manual PharMingen). Harvest recombinant virus 5 days post-transfection and amplify twice to obtain higher titered viral stocks.

     
  3. 3.

    Grow Sf9 insect cells at 28 °C in 1 L of Sf900-II serum-free medium in shaker flasks at 90 rpm.

     
  4. 4.

    Infect cells with the recombinant virus once they reach a density of 2 × 106 cells/mL at an MOI of 1.

     
  5. 5.

    Harvest 48 h postinfection by centrifugation at 6000 × g for 15 min.

     
  6. 6.

    Resuspend pellet in 50 mL of PBS and store at −80 °C.

     
  7. 7.

    We routinely expressed 10 L of cells.

     

3.5 Deuteration of Sx4(C141S) (Small Scale)

  1. 1.

    Transform pET20b-Sx4(C141S) into BL21(DE3)pLysS cells by heat-shock, plate onto minimal media agar, and incubate overnight at 37 °C.

     
  2. 2.

    Use a single colony picked from the agar plate to inoculate 100 mL M9 media made up in 50% 2H2O supplemented with 100 μg/mL ampicillin and incubate at 37 °C with shaking at 220 rpm until an OD600 of 1 is reached (see Note 13 ). An incubation time of approximately 48 h is required to reach an OD600 of 1.

     
  3. 3.

    Use cells grown in 50% 2H2O to inoculate 100 mL of fresh M9 media made up in 70% 2H2O at an OD600 of 0.1. Incubate at 37 °C until an OD600 of 1 is reached. An OD600 of 1 is generally achieved after an overnight incubation.

     
  4. 4.

    Repeat step 3, inoculating fresh M9 media made up in 90% 2H2O with the 70% culture and finally inoculating the fresh M9 media made up in 99% 2H2O with the 90% culture.

     
  5. 5.

    For protein expression, use cells from a culture of 99% 2H2O to inoculate 99% 2H2O minimal media to an OD600 of ~0.01 and incubate at 37 °C with shaking until an OD600 of 0.5–0.6 (approximately 2–3 h post-inoculation) is reached.

     
  6. 6.

    Induce protein expression by the addition of 1 mM IPTG to the cultures.

     
  7. 7.

    Allow protein expression to proceed for 24 h at 37 °C.

     
  8. 8.

    Harvest cells by centrifugation at 8000 × g for 10 min at 4 °C.

     
  9. 9.

    Freeze the harvested cells in liquid nitrogen and store at −80 °C.

     

3.6 Large-Scale Deuteration of Sx1 and ΔNSx1

All large-scale protein deuteration was conducted following the methods described in Chen et al. [35] and Duff et al. [36].
  1. 1.

    Transform pET24a-Sx1 or pET24a-ΔNSx1 plasmid into 50 μL of commercial competent BL21(DE3)* cells by the standard heat-shock method.

     
  2. 2.

    Add the transformation mixture to 250 μL SOC media and incubate for 1.5–2.0 h at 37 °C without shaking.

     
  3. 3.

    Add the SOC/transformation mixture to 10 mL of 50% 2H2O ModC1 media in a 125 or 250 mL flask and shake at 220 rpm at 37 °C for 16–20 h, until an OD600 value between 0.4 and 1.0 is reached.

     
  4. 4.

    Allow 0.7 mL for sampling and add the remaining 9.3 mL to ~40 mL of 100% 2H2O ModC1 in a 1 or 2 L flask. This results in a 90% 2H2O media.

     
  5. 5.

    Shake the flask at 220 rpm at 37 °C for two generations (the doubling time of BL21(DE3) cells in 90% v/v 2H2O is approximately 3 h). At ~6 h, dilute the culture into 102 mL by adding fresh 90% 2H2O ModC1 in a 2 L flask. Continue incubation at 220 rpm at 37 °C until an OD600 of 0.8–1.0 is reached.

     
  6. 6.

    Allowing 2 mL for sampling use 100 mL to inoculate a bioreactor containing 900 mL 90% 2H2O ModC1. Stir the bioreactor at a high speed, at 37 °C.

     
  7. 7.

    At a point between OD600 = 1 and 5, pH and foaming control is required. The pH, controlled by the addition of 28% NH4OH, is set to be higher than 6.2. Add 60–100 μL/L antifoam 204 to control foaming. Foaming behavior may vary depending on temperature, airflow, impellor speed, and impellor height with respect to the height of the media. Add additional antifoam 204 as required, at 60–100 μL/L.

     
  8. 8.

    Monitor the dissolved oxygen tension (DOT), and ensure that the culture remains aerobic throughout by increasing impeller speed to maximum, and then by lowering the culture temperature, which may be done automatically or with manual override. As the DOT may drop suddenly, action should be taken if and when the DOT drops to 80%. The culture temperature may also be lowered to prolong the time of induction (step 9) to be in working hours . Thus, the culture temperature before induction may be any temperature between 20 °C and 37 °C (see Note 14 ).

     
  9. 9.

    When an OD600 of 16 is approached (aim for 16 but no higher), drop the temperature to 20 °C and add IPTG to a final concentration of 1 mM (see Notes 15 and 16 ).

     
  10. 10.

    Harvest cells by centrifugation at exhaustion of the carbon source, as judged by the simultaneous rise in dissolved oxygen tension and pH.

     

3.7 Protein Purification

To inhibit degradation and maintain stability, all proteins and buffers should be kept as close as possible to 4 °C. This is done preferably by performing each step in a cold room or otherwise keeping everything on ice. Protease inhibitors, β-ME or DTT, and DNase at the concentrations detailed above in Subheading 2.4 should be added to buffers immediately prior to use.
  1. 1.

    Resuspend pellet in up to 200 mL cell lysis buffer as required per expressed L of pellet (see Note 17 ).

     
  2. 2.

    For bacterial cell pellets, resuspend vigorously using a syringe (10–50 mL) without a needle. BL21(DE3)pLysS generally lyses effectively when the frozen pellet is thawed. Rosetta2(DE3)pLysS cells are sonicated for 2 min 100% duty cycle, 40 Hz, repeated two times.

     
  3. 3.

    For insect cells, add His-Munc18c lysis buffer (50 mL/pellet from 1 L of culture), break pellet into as many small pieces as possible (we typically use a 10 mL plastic serological pipette), and then leave to thaw for 30 min. Homogenize gently with a 10 mL syringe (see Note 18 ).

     
  4. 4.

    Centrifuge lysis solutions at 42,000 × g at 4 °C for 30 min and retain supernatant.

     
  5. 5.

    Add affinity resin to the cell lysate (equilibrated in wash buffer 1 or cleavage buffer as appropriate) (see Notes 18 and 19 ).

     
  6. 6.

    Place resin/supernatant mixture into suitably sized centrifuge tube/s, attach to a rotary mixer or tube roller, and mix at 4 °C. Mixing for 2 h is generally sufficient but 15 min to overnight is possible depending on the resin and yield required (see Note 20).

     
  7. 7.

    When incubation is complete, pour resin suspension into a 20–30 mL disposable plastic gravity column (see Note 21 ).

     
  8. 8.

    Resuspend any residual resin remaining in the centrifuge tube/s with ~5 mL wash buffer. Repeat twice, pouring the resuspended resin into the gravity column.

     
  9. 9.

    Wash the resin bed in the gravity column with ~50 mL wash buffer 1, followed by ~50 mL of wash buffer 2. Minimal amounts of protein should be detected in the flow through by the end of wash buffer 2. Testing via Bradford Protein Assay (step 10) as well as SDS-PAGE is recommended to determine appropriate volumes of wash buffers used as well as whether buffer composition should be modified (see Note 22 ).

     
  10. 10.

    To test for protein in the wash buffer flow through, add 160 μL wash buffer flow through into 40 μL of Bradford assay dye reagent in a 96-well microplate. As a blank control, use 160 μL of a wash buffer in 40 μL of Bradford assay dye reagent. The wash step is complete when the flow through is of similar color intensity as the blank control (determined by eye).

     
  11. 11.

    When the washing step is complete, pipette ~3–6 mL elution buffer gently onto the resin bed, while aiming to minimize resuspension of the resin, to result in a few concentrated fractions rather than many dilute fractions. Collect ~1.0 mL fractions into 1.5 mL centrifuge tubes. To determine when protein elution is complete, pipette 40 μL of Bradford assay dye reagent into a 96-well microplate, 155 μL of distilled H2O, and 5 μL of eluted sample and mix thoroughly. As a blank control, add 160 μL water into a well containing 40 μL of Bradford assay dye reagent. When the sample is the same color as the blank control, the elution is complete.

     
  12. 12.

    Pool all protein fractions that exhibit a blue color in the Bradford assay and measure the total volume. If the pooled eluate volume is greater than 5 mL, concentrate to ~5 mL using an Amicon Ultra-15 centrifugal filter unit with a suitable MW cutoff (typically MW of protein divided by 3). Centrifuge at 3000 × g at 4 °C to desired volume/concentration. Be careful not to concentrate too far as this may cause protein to precipitate.

     
  13. 13.

    When the protein solution volume is ~5 mL the sample is ready to be purified further on a gel filtration column or anion-exchange column.

     
  14. 14.

    Run Munc18a-His over a Superdex 200 16/60 and His-Munc18c over a Superdex 200 26/60 column using size-exclusion buffer.

     
  15. 15.

    Inject syntaxin constructs onto MonoQ 5/50 column and elute with a salt gradient from 25 to 500 mM using MonoQ A and B buffers.

     
  16. 16.

    Pool fractions that gave the highest purity and concentrate using appropriate Amicon Ultra centrifugal filter units to the required concentration.

     

3.8 Preparation of Munc18c :Sx4 Complex by Mixing Lysates and Co-purification

Co-purification of Munc18c:Sx4 complex was achieved by following the protocol described by Hu et al. [33].
  1. 1.

    Resuspend cell pellets from 500 mL culture of Sx4 and 1 L culture of Munc18c protein expression in His-Munc18c lysis buffer using a syringe as described above in Subheading 3.7 (see Notes 23 and 24 ).

     
  2. 2.

    Mix the lysates together and incubate at 4 °C for 30 min. This is to allow the formation of the Munc18c:Sx4 protein complex.

     
  3. 3.

    Centrifuge the mixed lysates at 42,000 × g for 30 min at 4 °C.

     
  4. 4.

    Incubate the cleared lysate with TALON resin (1–2 mL/L of Sx pellet used) for 1.5–2 h.

     
  5. 5.

    Wash the resin beads with His-Munc18c wash buffers 1 and 2 (200 mL and 100 mL, respectively).

     
  6. 6.

    Wash beads with His-Munc18c cleavage buffer (20 mL) and resuspend in ~5 mL of cleavage buffer.

     
  7. 7.

    Incubate with 60 U of thrombin at room temperature for 2 h.

     
  8. 8.

    Add AEBSF to a concentration of 1 mM to end proteolysis.

     
  9. 9.

    Wash beads with His-Munc18c wash buffer to remove de-tagged Munc18 protein that is not complexed with Sx4.

     
  10. 10.

    Elute the Munc18c:Sx4 complex with Munc18 elution buffer.

     
  11. 11.

    Run the eluted protein on a Superdex 200 16/60 size-exclusion chromatography column equilibrated with Munc18 size exclusion buffer.

     
  12. 12.

    Pool peak fractions from the FPLC run and concentrate to desired volume/concentration (Fig. 2).

     
Fig. 2

Munc18:Sx complex preparation. An example of an FPLC elution profile from a Superdex 200 26/60 size-exclusion chromatography column. Peak fractions are collected and concentrated for the scattering experiments. Inset. SDS-PAGE image of the peak fractions (reproduced from [40])

3.9 Preparation of Munc18–Sx Complex by Mixing Purified Proteins

Both non-deuterated and deuterated Munc18a:Sx1 and Munc18a:ΔNSx1 complexes were prepared by mixing purified proteins as described below.
  1. 1.

    Purified proteins were mixed in a protein molar ratio of ~1:1.8 Munc18:Sx (see Note 25 , for examples of the quantity of protein to use). Protein mixture was incubated at 4 °C for 2 h on a rotary mixer and Munc18 size-exclusion buffer was added to bring the total volume up to required volume, for example 2 mL, before injection onto a Superdex 200 16/60 column.

     
  2. 2.

    Peak fractions from the FPLC run were pooled and concentrated to desired volume/concentration.

     

3.10 Preparation of Munc18c :Deuterated Sx4 (DSx)Complex

The protocol described above in Subheading 3.8 was used to prepare Munc18c:DSx4 complex with the following changes.
  1. 1.

    Increase the DSx:Munc18 cell pellet ratio, i.e., use pellet from 2 L of DSx4 culture and pellet from 1 L of Munc18c pellet (see Note 26 ).

     
  2. 2.

    Use 50 U of thrombin rather than 60 and reduce incubation time to 1 h.

     
  3. 3.

    Use 600 mM imidazole to elute the protein complex.

     

3.11 MALDI–TOF Mass Spectrometry to Determine Level of Deuteration

  1. 1.

    Desalt Sx and DSx using C18 Zip tips.

     
  2. 2.

    Spot 0.5 μL desalted proteins in 70% acetonitrile and 0.1% formic acid with 0.5 μL of sinapinic acid matrix onto a MALDI plate and allow to air-dry.

     
  3. 3.

    Collect mass spectrum using the Voyager DE.

     

3.12 Chemical Cross-Linking and Mass Spectroscopy

3.12.1 DTSSP Cross-Linking

  1. 1.

    Mix purified Munc18c:Sx4 complex (~1.2 μM) in 50 mM HEPES buffer pH 7.5 with 250 μM DTSSP for 4 min (see Notes 27 and 28 ).

     
  2. 2.

    Add Tris–HCl, pH 8.5, to a concentration of 0.1 M and allow the reaction to proceed for 10 min.

     
  3. 3.

    Carbamidomethylate the sample by bringing the solution to 50 μM in iodoacetamide and react for 30 min in the dark (see Note 29 ).

     

3.12.2 Sample Preparation

  1. 1.

    Add PAGE sample buffer without reducing agent to the solution and concentrate the sample to 20 μL using a 30 kDa molecular weight cutoff centrifugal concentrating device.

     
  2. 2.

    Run concentrated samples on a nonreducing SDS-PAGE gel and cut out bands for in-gel tryptic digestion.

     
  3. 3.

    Bands were dehydrated in 1 mL of 100% methanol for 5 min at room temperature.

     
  4. 4.

    Rehydrate gel pieces in 1 mL of 30% methanol for 5 min.

     
  5. 5.

    Wash gel pieces twice in water for 10 min each, followed by washing three times in 100 mM NH4CO3 containing 30% acetonitrile for 10 min.

     
  6. 6.

    Cut the gel into small pieces and dry in Alpha-RVC vacuum concentrator for 30 min.

     
  7. 7.

    Resuspend the dry gel pieces in 50 mM NH4CO3.

     
  8. 8.

    Add trypsin (1 μg/10 μg protein) to the solution and incubate overnight at 37 °C (see Note 30 ).

     
  9. 9.

    After the tryptic digestion is complete the samples are spun at maximum speed in a microcentrifuge for 1 min and the supernatant transferred to a microcentrifuge tube.

     
  10. 10.

    Serially extract peptides from the gel pieces using 3 × 50 μL of 50% acetonitrile containing 0.1% formic acid.

     
  11. 11.

    Combine the fractions with the supernatant and dry the sample using a vacuum concentrator.

     
  12. 12.

    Reconstitute samples in 50% acetonitrile and 0.1% formic acid.

     
  13. 13.

    For manual MALDI analysis, mix 0.5 μL of the reconstituted sample with 0.5 μL of the matrix and spot onto a MALDI plate.

     
  14. 14.

    Analyze sample using MALDI TOF /TOF or LC MALDI TOF/TOF mass spectrometry.

     

3.12.3 BS3 Cross-Linking

  1. 1.

    Similarly, incubate Munc18:Sx complexes (~2.0 μM) in BS3 reaction buffer with 62 μM iodoacetamide at room temperature for 30 min (see Note 31 ).

     
  2. 2.

    Add the cross-linking reagent BS3 to a concentration of 690 μM and allow the reaction to proceed for another 30 min.

     
  3. 3.

    Stop the reaction with the addition of 50 mM NH4HCO3 pH 8.0.

     

3.12.4 Sample Preparation

  1. 1.

    Concentrate the sample to 50 μM in a 30 kDa concentrator and transfer to a microcentrifuge tube.

     
  2. 2.

    Digest the cross-linked protein complex by adding 7.5 μg of trypsin.

     
  3. 3.

    Add 8 μL of working stock formaldehyde followed by 4 μL of 1 M dimethylamine-borane complex to 75 μL of the digested protein complex. Leave overnight at 4 °C for reductive methylation to occur (see Note 32 ).

     
  4. 4.

    Desalt the methylated peptides, and the native sample, using Zip tips and analyze by LC MALDI/TOF-TOF mass spectrometry or LC electrospray ionization mass spectrometry.

     
  5. 5.

    Cross-linked peptides were assigned by identifying the presence of the precursor m/z (the intact MH+), the partial peptide sequence of at least one of the peptides, and the presence of the a ions corresponding to the dimethylated N-terminal amino acids of both peptides [37] (see Note 33 ).

     

3.13 SAXS

  1. 1.

    Concentrate the purified Munc18:Sx protein complexes from Subheadings 3.8, 3.9, or 3.10 to approximately 3 mg/mL (see Note 34 ).

     
  2. 2.

    Make at least two serial dilutions of the stock solution (see Note 35).

     
  3. 3.

    Determine the absorbance of each solution at 280 nm, and determine the concentration using the theoretical extinction coefficients (Table 2).

     
  4. 4.

    Measure SAXS data from the buffer first, followed by the protein solutions, starting from the lowest concentration to the highest. For proteins of this size a q-range of ~0.01–0.40 Å−1 is appropriate (see Note 36 ).

     
  5. 5.

    Average and reduce data to 1-dimensional profiles, making appropriate corrections for sample transmission, background radiation, and detector efficiency. Then subtract the solvent scattering from the protein + solvent scattering to give the scattering profile from the protein alone.

     
Table 2

Protein parameters used to calculate protein concentration

Protein

Theoretical extinction coefficient (M−1 cm−1)

Molecular weight (Da)

Munc18a

61,770

67,568.71

Munc18c

68,300

67,942.49

Sx11–261

7,450

30,182.76

Sx41–275

4,470

31,929.09

Munc18a- Sx11–261

69,220

97,733.45

Munc18c- Sx41–275

72,770

99,853.57

Values were calculated using the ProtParam Tool [41] (https://web.expasy.org/protparam/)

3.14 Neutron Scattering

  1. 1.

    Concentrate the purified protein solution to approximately 3 mg/mL (see Note 34 ).

     
  2. 2.

    Make up appropriate buffer solutions by mixing the SANS 1H2O and SANS 2H2O buffers together at appropriate ratios. Here, the two buffer solutions were 40% 2H2O and 100% 2H2O (see Note 37 ).

     
  3. 3.

    Inject ~350 μL of protein solution (see Note 38 ) into a number of dialysis cassettes, place each cassette into 50–100 mL of one of the prepared SANS buffers (see Note 39 ), and dialyze these overnight with gentle rocking if possible.

     
  4. 4.

    Retrieve the protein solutions from the dialysis cassettes, and take an aliquot of each dialysate (buffer). Centrifuge at >10,000 × g to remove any large particles from the solution.

     
  5. 5.

    Load the samples and buffers into the quartz cells and measure SANS data between a q-range of ~0.01 and 0.40 Å−1.

     
  6. 6.

    Average and reduce data to 1-dimensional profiles , making appropriate corrections for sample transmission, background radiation, and detector efficiency. Then subtract the solvent scattering from the protein + solvent scattering to give the scattering profile from the protein alone for each contrast point.

     

3.15 Data Analysis

  1. 1.

    Determine masses from I(0), and ensure that these are as expected based on the composition of the sample.

     
  2. 2.

    For the SANS data, analyze the dependence of Rg upon contrast using a Stuhrmann plot (a plot of Rg2 vs. Δρ−1 that should be parabolic in shape) or the parallel axis theorem. These analyses will give an indication of the Rg of each component of the complex, and the arrangement of the subunits.

     
  3. 3.

    Use high-resolution structures of the components for rigid-body modeling. These can be crystal structures and/or homology models. Due to the number of missing residues in the crystal structures of Munc18 and Sx proteins, iTasser [38] was used to generate high-resolution models based on the crystal structures (see Note 40 ).

     
  4. 4.

    Define the rigid domains in the complex. For this work, the Munc18 proteins were defined as two rigid subunits (Domain 1, and combined Domains 2/3a/3b). Syntaxin proteins were defined as four rigid subunits (N-peptide, linker region, Habc domain, and H3 domain). The relationship between Munc18 Domain 1 and the Sx N-peptide was fixed in the same conformation as that observed in the crystal structures of Munc18 proteins with the N-peptide [10, 11].

     
  5. 5.

    Set up distance restraints. Here, distance restraints were applied to preserve the connectivity of each polypeptide chain (i.e., for Munc18 proteins the last Cα in Domain 1 was restrained to lie within 3.8 Å of the first Cα in Domain 2, and likewise for syntaxin). Additionally, distance restraints were defined between residues identified in the cross-linking analysis. A restraint distance of 35 Å between Cα atoms was used for BS3 and DTSSP cross-links. This is significantly longer than what would be expected for these cross-linkers, but allows leeway for cross-links that may have been formed between mobile and dynamic regions.

     
  6. 6.

    Run the rigid-body modeling procedure against all datasets simultaneously. In this work SASREF 7 [29] was used to optimize Munc18:Sx models against SAXS data (looking at the entire complex), and two SANS datasets (40% 2H2O—looking at the Sx component; 100% 2H2O—looking at the Munc18 component). The quality of the model is taken from both the fit to the scattering data and whether the model satisfies the distance restraints placed on it.

     

4 Notes

  1. 1.

    Prepare the M9 salts (disodium hydrogen phosphate anhydrous, potassium dihydrogen phosphate, sodium chloride, ammonium chloride) separately in the appropriate 1H2O:2H2O ratios. Prepare two solutions of glucose, magnesium sulfate, calcium chloride, thiamine, and iron (II) sulfate, one in 1H2O, and the other in 2H2O, and filter sterilize. Mix them in the appropriate ratios to prepare 50% 2H2O, 70% 2H2O, 90% 2H2O, or 99% 2H2O. Mix 50 mL of the glucose solution with 950 mL of M9 salts prior to adding in the bacterial cultures.

     
  2. 2.

    The specific bioreactor is not important. Any bioreactor that allows for controlling the temperature, stirring speed, and air supply; pH monitoring; base feed for pH control; and dissolved oxygen tension monitoring can be used.

     
  3. 3.

    The complete media is not soluble, so the preparation is divided into a soluble, stable, “base solution,” and four additives that are added in a concentrated form. After addition of additives A, B, C, and D to base media, a fine precipitate will form. For this reason, A, B, C, and D are added only just before use. Mixing of the additive stock solutions will also result in precipitation and as such care must be taken not to cross-contaminate (especially A and B).

     
  4. 4.

    DNase from Roche was specifically used as we found that other brands could clip a loop in M18a, which did not seem to affect the protein structurally but was obvious on a denaturing gel when run beside an unclipped M18a.

     
  5. 5.

    β-ME should be added to the buffer immediately before use from a 1 M stock that is less than 1 week old. β-ME has a half-life of 4 h at pH 8.5.

     
  6. 6.

    A thermal stability assay conducted on Sx4 indicated that this protein may be more stable in a TAPS buffer solution. However, as Tris is used extensively in the lab for other proteins, Tris is normally used to reduce the number of buffer solutions that need to be prepared.

     
  7. 7.

    The pH of Tris can fluctuate by as much as 0.5 pH units as the temperature of the buffer solution moves from 4 °C to 25 °C. The pH of the buffer should be adjusted at the temperature at which it will be used (generally 4 °C).

     
  8. 8.

    When using a H+ probe on a conventional pH meter, the actual pD of the solution is approximately pHmeasured −0.4; hence, the D2O SANS buffer has a different pH to the H2O SANS buffer.

     
  9. 9.

    Glycerol stocks were generally used for convenience and prepared by mixing 0.6 mL of an overnight culture with 0.4 mL 80% sterile glycerol and flash freezing at −80 °C. In the protocol, 10 μL refers to a stab into the culture and removal of 10 μL of frozen stock.

     
  10. 10.

    Measuring the OD at 600 nm at a dilution of ~1:100 (10 μL of culture in 990 μL of water) ensures that absorbance is less than 0.4, and thus proportional to the cell mass in solution. Absorbance readings should be corrected by the dilution factor.

     
  11. 11.

    Plasmids were transformed into the respective chemically competent E. coli strains. Fresh transformations worked better in terms of protein yield but this takes longer. A streak through the plate was often used to grow the starter culture rather than a single colony as variation was seen in how well single colonies expressed protein.

     
  12. 12.

    This protocol was developed to address issues relating to variable cell growth and expression. This may have been caused by phosphate in the rich media inhibiting the kanamycin resistance as described in [34].

     
  13. 13.

    Although 100 mL media was used in these experiments, any volume that would give an adequate amount of cells to inoculate the next batch of media in the sequence at an OD600 of 0.1 would suffice.

     
  14. 14.
    If the culture is healthy and adapted to the current deuterated media, the doubling time of the culture, g, should be constant. The value of g should be measured regularly to ensure exponential growth. Given g, the time until induction is given by
    $$ \mathrm{Time}\ \mathrm{until}\ \mathrm{induction}=g\times {\log}_2\left(\frac{{\mathrm{OD}}_{\mathrm{induced}}}{{\mathrm{OD}}_{\mathrm{current}}}\right) $$
    (3)
    where ODinduced is the target OD for induction (usually 16), and ODcurrent is the current OD. The culture temperature may be varied so as to adjust the predicted time of induction, as g varies predictably with temperature. We observe that the general Arrhenius relationship between chemical/biochemical activity and temperature holds for uninduced exponentially growing E. coli in deuterated ModC1 [36]. Accordingly, the generation time follows the equation
    $$ g(T)={g}_{37{}^{\circ}\mathrm{C}}\times {2}^{\frac{37^{{}^{\circ}}\mathrm{C}-T}{10^{{}^{\circ}}\mathrm{C}}} $$
    (4)
    where g(T) is the generation time at temperature T. Alternatively, to calculate the temperature required to obtain a desired doubling time (i.e., a doubling time that yields a convenient induction time), Eq. (4) can be expressed as
    $$ T(g)={37}^{{}^{\circ}}\mathrm{C}-{10}^{{}^{\circ}}\mathrm{C}\times {\log}_2\left(\frac{g}{g_{37{}^{\circ}\mathrm{C}}}\right) $$
    (5)
     
  15. 15.

    Early deuterated production runs used 5 mM IPTG , but this was found to be unnecessarily high.

     
  16. 16.

    Different postinduction culture temperatures were used in early productions. In initial productions, the postinduction culture temperature was set at 30 °C, but, due to bioreactor aeration limitations, this was found, by analysis of the bioreactor data logs, to be incompatible with maintaining aerobic conditions throughout (these productions endured oscillations (~0.1 Hz) in DOT and temperature, we considered this undesirable). In later deuterated productions a postinduction temperature of 15 °C was tested. In all subsequent productions, the postinduction temperature was 20 °C. The lower incubation temperatures enable aerobic conditions to be comfortably maintained; lower temperatures lengthen the production time with no observed benefit to protein yield or quality.

     
  17. 17.

    Ratio of lysis buffer to pellet volume is important to achieve maximum yield of protein. This should be tested for each protein. However, we found that most proteins gave a good yield using 100–200 mL of buffer per expressed L of pellet. This also takes into account the amount of protein expressed and the equipment required downstream to process, e.g., centrifuges.

     
  18. 18.

    We found that Munc18c expressed in insect cells is very susceptible to aggregation when shear force is applied or by other mechanisms—until the protein is bound on resin. Care needs to be taken at the lysis step to lyse the cells gently with minimal shear force. We syringe the lysis mixture up and down very gently to break the cells—think of operating the syringe as if it were a very fragile glass rod.

     
  19. 19.

    Generally, 1–3 mL of any IMAC resin per liter of pellet but this should be determined experimentally as each protein and resin has different binding properties.

     
  20. 20.

    Both IMAC batch and column purification methods are possible although this protocol is written for batch. To incubate, place the supernatant and resin into a suitably sized container, i.e., 50 mL tube or 500 mL tube as volume allows, and roll at a suitable speed to stop sedimentation.

     
  21. 21.

    We found that His-Munc18c lysate from insect cell expression restricted the flow through the purification resin. Pouring the resin and supernatant suspension directly into the disposable column could potentially maximize yield by limiting resin loss due to handling, but this causes the resin to block requiring constant manual resuspension in the column, thereby increasing processing time. A faster solution was to allow the resin to settle first (usually by centrifuging at a sufficient speed to pellet the resin and not pellet other particulates, i.e., 500 rpm) and then carefully pouring off most of the supernatant ensuring that the pelleted resin is not resuspended. The remaining supernatant in the tube is then used to resuspend the pelleted resin and this suspension is then poured into the column.

     
  22. 22.

    Washing times and therefore the amount of wash buffer required varied depending on the amount of protein purified. The standard rule of thumb we used was 20× resin bed volume, i.e., 5 mL bed volume required 100 mL of total wash buffer. However total volume and stringency (increasing salt concentration up to 1 M or imidazole concentration up to 50 mM) of wash buffer can be modified to improve purity if required.

     
  23. 23.

    Sx4 constructs can be lysed in His-Munc18c lysis buffer with no detrimental effects.

     
  24. 24.

    The amount of cell pellet used is based on the relative expression levels of the two proteins and is chosen so that there is an excess of Munc18c. This ensures that all of the Sx4 forms a complex with Munc18c. Unbound Munc18c is removed as the flow through upon rebinding of sample to metal affinity resin after thrombin cleavage.

     
  25. 25.

    Examples of ratios used: Munc18a 2.8 mg (780 μL of 3.6 mg/mL) plus Sx1 2.3 mg (320 μL of 7.3 mg/mL) gave 1.6 mg of a final purified 1:1 complex. Munc18a 1.4 mg (1 mL of 1.4 mg/mL) plus 1.1 mg ΔNSx1 (160 μL of 6.9 mg/mL) gave 1.5 mg of a final purified 1:1 complex.

     
  26. 26.

    DSx4 is less stable and less soluble than its non-deuterated counterpart.

     
  27. 27.

    DTSSP is a cleavable cross-linker whose disulfide bond fragments in MALDI TOF-TOF mass spectrometry to give a distinctive 66-m/z pair corresponding to the asymmetric fragmentation of the disulfide bond.

     
  28. 28.

    For lysine-preferring cross-linkers such as DTSSP a pH in the range of 7–9 is ideal and conditions under which complex formation is first observed are preferable for lengthy reactions. Optimal conditions for cross-linking are established using PAGE electrophoresis.

     
  29. 29.

    Carbamidomethylation by reaction with iodoacetamide was used with or without reduction to prevent the adventitious formation of disulfides or to reduce native disulfides as disulfides can make peptides too large for ready detection by mass spectroscopy.

     
  30. 30.

    Trypsin is the protease of choice for use in this work; however other proteases are also suitable in specific cases. In-gel digestion while reducing peptide yield can ensure that specific products are digested; solution digest of cross-linked proteins produced in defined circumstances increases yield of peptides.

     
  31. 31.

    BS3 is a stable cross-linker free of the possibility of disulfide-exchange reactions.

     
  32. 32.

    Reductive dimethylation of peptides using dimethylamino borane and formaldehyde was used as an aid in the identification of cross-linked peptides as cross-linked peptides should produce two a-ions in the MS fragmentation spectra corresponding to both amino termini.

     
  33. 33.

    For Munc18c:Sx4 complexes both DTSSP and BS3 in combination with reductive alkylation were used to successfully identify cross-linked peptides. However, we found that the use of BS3 in combination with the use of XQuest [39] to search the MS data was the most effective approach. Therefore we only used BS3 with reductive alkylation for cross-linking the Munc18a:Sx1 complex. Information derived from the BS3 cross-linking experiments was used in the rigid-body modeling experiments.

     
  34. 34.

    Concentrator (Amicon Ultra centrifugal filter unit) should be rinsed with water or buffer before use. The centrifugation step should not be rushed by using high speeds; we used centrifugation speeds of ~3000 × g and runs of no longer than 5–10 min. We also mixed the protein solution in the concentrator by pipetting gently between each centrifugation step.

     
  35. 35.

    Alternatively, the concentrated protein can be dialyzed against a buffer, and the dialysate used to dilute the protein.

     
  36. 36.

    It is preferable to measure the buffer scattering before and after measurement of the scattering from the protein solutions to ensure that no material was deposited on the sample capillary. Collection times vary depending on the instrumentation used (hours for lab-based instruments, to seconds for synchrotron sources).

     
  37. 37.

    Unlabeled proteins were contrast matched in approximately a 40% v/v 2H2O buffer (i.e., four parts 2H2O SANS buffer to six parts 1H2O SANS buffer), while 2H-labeled proteins were matched out at a much higher level (i.e., for a protein deuterated to a level of ~75%, the contrast match point is close to 100% 2H2O buffer).

     
  38. 38.

    The nominal volume of a 1 mm QS-120 cell is 280 μL, so that working with 350 μL (or more) allows a cell to be filled comfortably given the losses that invariably occur during unloading of the dialysis cassette. Further, when working with buffers with high concentrations of 2H2O, it is technically feasible to use 2 mm QS-120 cells with a nominal volume of 560 μL, so that working with at least 650 μL will allow a 2 mm cell to be filled comfortably.

     
  39. 39.

    Dialysis cassettes are convenient for the small volumes dialyzed, but tubing or any number of commercial dialysis devices can also be utilized. Further, in general a buffer change during dialysis is not generally required, as the exact deuterium content is less important than the sample and dialysate being completely exchanged.

     
  40. 40.

    Rigid-body modeling was utilized as the primary modeling method as cross-link restraints cannot be applied to an ab initio model.

     

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andrew E. Whitten
    • 1
  • Russell J. Jarrott
    • 2
  • Shu-Hong Hu
    • 2
  • Anthony P. Duff
    • 1
  • Gordon J. King
    • 3
  • Jennifer L. Martin
    • 2
  • Michelle P. Christie
    • 4
    Email author
  1. 1.Australian Nuclear Science and Technology OrganisationLucas HeightsNSWAustralia
  2. 2.Griffith Institute for Drug DiscoveryGriffith UniversityNathanAustralia
  3. 3.Centre for Microscopy and MicroanalysisThe University of QueenslandSt LuciaAustralia
  4. 4.Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia

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