Immobilization of lipid nanorods onto two-dimensional crystals of protein tamavidin 2 for high-speed atomic force microscopy

Summary High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action. For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1


Note:
The mica disc can be removed using acetone.

KEY RESOURCES TABLE
Figure 1.The method to glue a mica disc on a glass stage It is recommended to perform these under a stereo microscope.

MATERIALS AND EQUIPMENT
Ampicillin (100 mg/mL) Add deionized distilled (ddH 2 O) to 1.00 g of ampicillin up to 10 mL (total volume).Vortex until ampicillin is completely dissolved.Filter the solution through a 0.2-mm syringe filter.Dispense the solution into 1.0-mL aliquots.Ampicillin (100 mg/mL) can be stored at À20 C for up to 1 year.Crystal buffer (20 mM of HEPES-NaOH containing 400 mM of NaCl, 200 mM of MgCl 2 and 20% PEG6000, pH 8.5) Add 4 mL of Buffer A to 1.0 g of PEG6000 in a 50 mL centrifuge tube.Vortex until PEG6000 is completely dissolved.The final volume will be $5 mL.Crystal buffer can be stored at $23 C for up to 1 year.
Buffer A (25 mM of HEPES-NaOH containing 500 mM of NaCl and 250 mM of MgCl 2 , pH 8.5)

Final concentration Amount
NaCl This section describes the preparation of tamavidin 2 protein, lipid nanorods and tamavidin 2 2D crystals covering the mica surface, as well as the immobilization of lipid nanorods on the 2D crystals.Tamavidin 2 is an avidin-like biotin-binding protein from Pleurotus cornucopiae, and it consists of 141 amino acid residues.The article by Takakura et al. 3 contains detailed information on the discovery, properties, expression, and purification methods of tamavidin 2. The sequence of tamavidin 2 is available from GenBank (GenBank: AB102785) (Figure 2).

Prepare the expression vector for production of tamavidin 2 protein
Timing: 2-4 weeks 1.Prepare an appropriate plasmid for protein expressions in Escherichia coli (E.coli) cells.
Note: Expression vector pET-17b (Novagen) with the DNA sequence coding tamavidin 2 in NdeI-XhoI sites (pET-17b-tamavidin 2) in our case was designed and purchased from GenScript.

Timing: 4 days
This subsection summarizes our modified method of expression and purification of tamavidin 2 protein from the report by Takakura et al. 3 Tamavidin 2 is expressed at high levels in soluble fractions in recombinant E. coli and can be purified in a single step using a 2-iminobiotin agarose gel.
3. Grow the E. coli cells harboring the expression vector in LB medium (800 mL) containing an appropriate antibiotic (e.g., 100 mg/mL of ampicillin for pET-17b vector) at 37 C until the absorbance at 600 nm reaches 0.5-0.8. 4. Add 800 mL of 0.5 M IPTG (final concentration: 0.5 mM) to the LB medium.Shake at $120 rpm for $18 h at 25 C. 5. Harvest the cells by centrifugation at 4 C (4000 3 g for 10 min).6. Store the cell pellet at À80 C. 7. Prepare Buffer L, Buffer W, Buffer E, and Buffer S. See the 'materials and equipment' section for preparation.8. Purify tamavidin 2 following the protocol by Takakura et al. 3 Below is the protocol with the slight modification.a. Resuspend the pellet in Buffer L (50 mL) and add 50 mL of 1 M phenylmethylsulfonyl fluoride (PMSF) (final concentration: 1 mM).b.Sonicate the suspension on ice (0.2 s of pulse with an interval of 0.8 s at $200 W for 10 min).c.Centrifuge the suspension at 4 C (18,000 3 g for 40 min) and collect the supernatant.d.Purify the clear supernatant using 2-iminobiotin agarose (Thermo Fischer Scientific).
ii. Equilibrate the column with 4-column volumes of Buffer L. iii.Apply the supernatant to the column.iv.Wash the column with 20-column volumes of Buffer W. v. Elute tamavidin 2 protein by adding 5-column volumes of Buffer E. vi.Concentrate the eluted tamavidin 2 solution and exchange buffer to Buffer S using Amicon Ultra-15 (NMWL 10k).vii.Centrifuge the tamavidin 2 solution (12,000 3 g at 4 C for 10 min) and collect the supernatant.
Note: Stability of tamavidin 2 is very high and its biotin-binding activity is rarely reduced by storage in solution at 4 C for at least 1 year. 3In our experiment, tamavidin 2 solution purified and stored at 4 C for >3 years is used.

Prepare lipid nanorods
Timing: R3 h Lipid nanorods, formed by the spontaneous assembly of galactocerebrosides, have been used for HS-AFM observation of dynamin. 4Biotinylated lipids must be included to immobilize lipid nanorods onto tamavidin 2 2D crystals.Below is our slightly modified protocol from the report by Colom et al. 4 9. Prepare a 100-nmol mixture of phospholipids containing galactosylceramide and biotinyl cap PE (in our case 1 : 40 mL of 1 mM galactosylceramide; 15 mL of 1 mM POPC; 5 mL of 1 mM POPE; 6 mL of 1 mM PI; 6 mL of 1 mM POPS; 6 mL of 1 mM POPA; 12 mL of 1 mM cardiolipin (CL); 10 mL of 1 mM biotinyl cap PE [40:15:5:6:6:6:12:10 mol %] in chloroform (100 mL in total)) in a 5-mL glass bottle.10.Dry chloroform under a gentle stream of nitrogen gas to produce lipid film.11.Place the glass bottle in a vacuum desiccator for R2 h to completely evaporate the chloroform.
Note: You can keep the glass bottle in a vacuum desiccator for 24 h.12. Add 100 mL of buffer you want to use (e.g., 20 mM of HEPES-NaOH containing 150 mM of NaCl, pH 7.0) to the lipid film and incubate for 10 min followed by vortexing to prepare 1 mM lipid mixture solution.13.Transfer the lipid mixture solution to a 1.5-mL microcentrifuge tube.14.Sonicate it in a bath sonicator for 10 min (40 W, 42 kHz).15.Sonicate it further with a tip sonicator for %5 s (50 W, 20 kHz).
CRITICAL: Longer sonication time produces vesicular structures rather than nanorods.Details are shown in 'problem 5' of 'troubleshooting' section.
16. Protect from light and keep the 1.5-mL tube on ice until use.This subsection describes the preparation of tamavidin 2 2D crystals on mica.Tamavidin 2 2D crystals have been a useful substrate that develop directly on the mica surface to immobilize biotinylated proteins or membranes. 5,6See the protocol by Uchihashi et al. 2 for HS-AFM setup and observation.We use a sample scanning HS-AFM instrument (MS-NEX, Research Institute of Biomolecule Metrology Co., Ltd.) and cantilevers (length: $7-mm, width: $2-mm, thickness: $0.08-mm, resonant frequency: 1.2 MHz in air, spring constant: 0.15 N/m) with electron beam deposited/EBD tips (USC-F1.2-k0.15,NanoWorld).HS-AFM images were viewed and analyzed using the software Kodec4.4.7.39. 7.Prepare crystal buffer and fixation buffer.See the 'materials and equipment' section for preparation.18. Add 1 mL of 150 mM of tamavidin 2 (A280 = 6.26) to 20 mL of crystal buffer (final concentration, 7.1 mM) and mix by gentle pipetting.
CRITICAL: Do not incubate for >60 min after the dilution with crystal buffer.Details are shown in 'problem 2' of 'troubleshooting' section.
19. Deposit a drop ($2 mL) of the diluted tamavidin 2 solution onto freshly cleaved mica by scotch tape and incubate for 5 min.20.Rinse the mica surface with 0.01% glutaraldehyde diluted with fixation buffer and keep the last drop on the mica for 5 min.
CRITICAL: Glutaraldehyde fixation after incubation of tamavidin 2 on mica is an indispensable step for stabilization of 2D crystals.Rinse with imaging buffer before glutaraldehyde fixation may wash out tamavidin 2 from the surface.
21. Rinse the mica surface with imaging buffer you want to use for HS-AFM imaging (in our case, 1 20 mM of HEPES-NaOH containing 150 mM of NaCl, pH of 7.0).22. Set up HS-AFM, start imaging and confirm 2D crystals of tamavidin 2 formed on the mica surface.
Note: Figure 3 shows an example of the expected results.
23. Keep a drop of imaging buffer on the mica after HS-AFM imaging.

Note:
The tamavidin 2 2D crystals covering the mica can be stored at $23 C in a humid container for several days.Figure 4 shows an example of a humid container.

Immobilization of lipid nanorods onto tamavidin 2 2D crystals
Timing: <10 min Immobilization of lipid nanorods can be achieved by applying the lipid nanorod solution to the tamavidin 2 2D crystals.
24. Mix 9 mL of buffer solution (e.g., 20 mM HEPES-NaOH containing 150 mM NaCl, pH 7.0) and 1 mL of lipid solution (kept on ice after sonication) to prepare 0.1 mM of lipid solution.25.Exchange the buffer solution on tamavidin 2 2D crystals to 0.1 mM of lipid solution.28.Apply proteins of interest to the lipid nanorods immobilized on tamavidin 2 2D crystals after removing the glass stage from the HS-AFM setup or add proteins of interest to the imaging buffer while HS-AFM imaging.
Note: You can incubate the proteins with lipid nanorods before applying it to tamavidin 2 2D crystals.The timing of addition of the proteins will depend on the proteins and the events you want to observe.For example, if you attempt to observe dynamin-coated lipid nanotubes, it would be preferable to mix dynamin and nanorods before fixation on tamavidin 2 2D crystals because fixation may interfere with the formation of dynamin assembly around the nanorods. 4

EXPECTED OUTCOMES
Immobilized lipid nanorods on tamavidin 2 2D crystals are the expected outcomes from this protocol (Figure 5).The lipid nanorods could be useful, especially for observation of proteins sensing membrane curvature such as proteins with BAR (Bin/Amphiphysin/Rvs-homology) domains. 8Nanorods are rigid enough to tolerate tapping force of the HS-AFM imaging.This protocol is an easy method  to prepare immobilized lipid rod structures for HS-AFM observation once sufficient amounts of purified tamavidin 2 proteins are obtained.

LIMITATIONS
The lipid nanorods formed by this protocol are rigid compared to those without galactosylceramide.Therefore, the dynamic processes of membranes, such as membrane fission, may be difficult to be observed. 4In addition, not only nanorods but also vesicular structures or layer structures could be observed on tamavidin 2 2D structures, as shown in Figure 5.You may need to select the region where nanorods with the appropriate length for your experiment are separated from other lipid assemblies.

TROUBLESHOOTING Problem 1
It is important to keep the mica surface wet to prevent binding of contaminants throughout the experiment after deposition of the tamavidin 2 solution on the mica (Step 20).However, it is difficult to exchange buffers (wash) with keeping the surface wet.

Potential solution
Removing the previous solution on mica should be done at the same time as the addition of solution to be replaced (Figure 6).Because larger micropipettes are difficult to push out solutions little by little, use of micropipettes for 2-20 mL volume is recommended.after bath sonication.Vesicular structures bound to the tamavidin 2 2D crystals can change to planar structures.Basically, tip sonication %5 s would be sufficient.

RESOURCE AVAILABILITY
Lead contact Further inquiries and requests for materials should be directed to the lead contact, Nobuo N Noda (nn@igm.hokudai.ac.jp).

Materials availability
All requests for resources and reagents are available from the lead contact, subject to a Materials Transfer Agreement.
-b-D-thiogalactopyranoside (IPTG) (0.5 M) Add ddH 2 O to 1.19 g of IPTG up to 10 mL (total volume).Vortex until IPTG is completely dissolved.Filter the solution through a 0.2-mm syringe filter.Dispense the solution into 1.0-mL aliquots.IPTG (0.5 M) can be stored at À20 C for up to 1 year.Phenylmethylsulfonyl fluoride (PMSF) (1 M)Add dimethyl sulfoxide (DMSO) to 1.74 g of PMSF up to 10 mL (total volume).Vortex until PMSF is completely dissolved.Dispense the solution into 50-mL aliquots.PMSF (1 M) can be stored at À20 C for up to 1 year.NaCl (1 M) Dissolve 58.4 g of NaCl in $700 mL of ddH 2 O. Add ddH 2 O up to 1000 mL.Filter the solution through a 0.22-mm filter membrane.NaCl (1 M) can be stored at $23 C for up to 1 year.
Filter the mixed solution through a 0.22-mm filter membrane.Fixation buffer can be stored at $23 C for up to 1 year.

Protocol 26 .
Keep a drop of 0.1 mM of lipid solution on mica for 5 min.27.Set up HS-AFM and start imaging.Note: Figure 5 shows an example of the expected results.Typically 3-10 lipid nanotubes with different length ($0.1-1 mm) are observed in 1 mm 3 1 mm area.

Figure 4 .
Figure 4. Photograph of a humid container example for keeping tamavidin 2 2D crystals on mica glued to a glass stage A sheet of Kimwipes is fixed with scotch tape and wetted with Milli-Q water in a plastic Petri dish.A drop of buffer solution (e.g., 20 mM HEPES-NaOH containing 150 mM NaCl, pH 7.0) ($2 mL) is kept on mica.The glass stage is loosely fixed to the bottom of the Petri dish with double-sided adhesive tape to prevent it from falling over.The space between the container and the lid is sealed with parafilm.

Figure 6 .
Figure 6.How to exchange buffer solutions on mica 20 mL of the solution to be replaced is pushed out from a micropipette (for 2-20 mL volume) on mica and absorbed with a twisted piece of Kimwipe simultaneously to keep the surface wet.It would be preferable to repeat this at least twice (R2 3 20 mL).
N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11.0 (0.2 M) Dissolve 4.43 g of CAPS in $70 mL of ddH 2 O and adjust pH to 11.0 with 1 N of NaOH ($13.2 mL).Buffer S (40 mM of potassium phosphate buffer, pH 7.0) Add 40 mM of KH 2 PO 4 ($40 mL) to 50 mL of 40 mM of K 2 HPO 4 until pH reaches 7.0.Filter the mixed solution through a 0.22-mm filter membrane.Buffer S can be stored at 4 C for up to 1 year.HEPES-NaOH, pH 8.5 (0.1 M) Dissolve 2.38 g of HEPES in $70 mL of ddH 2 O and adjust pH to 8.5 with 1 N of NaOH ($8.2 mL).Add ddH 2 O up to 100 mL.Filter the solution through a 0.22-mm filter membrane.HEPES-NaOH (0.1 M, pH 8.5) can be stored at $23 C for up to 1 year.MgCl 2 (2.5 M) Dissolve 50.8 g of MgCl 2 $6H 2 O in $70 mL of ddH 2 O. Add ddH 2 O up to 100 mL.Filter the solution through a 0.22-mm filter membrane.MgCl 2 (2.5 M) can be stored at $23 C for up to 1 year.
Filter the mixed solution through a 0.22-mm filter membrane.Buffer A can be stored at $23 C for up to 1 year.