Supplementary Methods

Additional materials

Reagents

BCA (bicinchoninic acid) protein assay kit (Pierce)

CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) (Sigma), 10% (w/v)

Chloramphenicol (Sigma), 34 mg/ml stock solution in ethanol

CymaL-6 and Cymal-7, 10% (w/v) (Anatrace),

Imidazole, for molecular biology, minimum 99% (Sigma)

Isopropyl -d-thiogalactopyranoside (IPTG) (Saveen), 1 M solution, filter sterilized

Kanamycin monosulfate (Sigma), 50 mg/ml stock solution, filter sterilized

Luria-Bertani Broth (LB medium) (Difco)

MgCl2, 1 M solution

N,N-dimethyldodecylamine N-oxide (lauryldimethylamine-oxide; LDAO) (Fluka), 10% (w/v)

n-decyl--d-maltopyranoside (DM) (Anatrace), 20% (w/v)

n-dodecyl--d-maltopyranoside (DDM) (Anatrace), 20% (w/v)

Ni-NTA Superflow (Qiagen)

n-octyl--d-glucopyranoside (Anatrace), 20% (w/v)

n-undecyl--d-maltopyranoside (UDM) (Anatrace), 20% (w/v)

PefablocSC (Biomol)

Phosphate-buffered saline (PBS): 1.44 g Na2HPO4*2H2O (8.1 mM phosphate), 0.25 g KH2PO4 (1.9 mM phosphate), 8.00 g NaCl, 0.2 g KCl in 1,000 ml H20; adjust pH to 7.4 using 1 M NaOH or 1 M HCl

Triton X-100 (Sigma), 20% (v/v)

Supplementary Protocol 1. Making CaCl2 competent cells and transformation.

1|Select the appropriate strain (see Supplemental table 2) and use 4-5 fresh colonies from a LB agar plate to inoculate 100 ml of LB medium (in the case of pLysS, include the antibiotic chloramphenicol (34 µg/ml)) in a 250 ml conical flask.

2|Incubate the culture at 37°C, with shaking at 220 r.p.m.

3|When the culture has reached an OD600 of approx. 0.5 (after 2-3 h), transfer it to two 50 ml Falcon tubes.

4|Cool the culture on ice for 10 min.

5|Centrifuge the cells at 2,600g at 4°C for 10 min.

6|Decant the supernatant.

7|Resuspend the cell pellet gently in 10 ml of ice-cold 0.1 M CaCl2.

8|Incubate resuspended cells on ice for 10 min.

9|Centrifuge the cells at 2,600g at 4°C for 10 min.

10|Decant the supernatant.

11|Resuspend the cell pellet gently in 2 ml of ice-cold 0.1 M CaCl2 plus 20% glycerol.

12|Incubate the resuspended cells on ice for 10 min.

13|Deliver 100 µl aliquots for future use (these competent cells can be used directly for a transformation: go to Step 16).

14|Snap freeze aliquots in liquid nitrogen and store at –80°C (competent cells can be stored at –80 °Cfor at least 6 months).

15|Thaw frozen competent cells on ice for 5-10 min before mixing the cells with DNA.

16|To transform the expression vector containing the membrane protein GFP-fusion (see supplementary Figure 1 and Supplementary Table 1) add 0.5 µl of expression vector (0.25 µg/µl of DNA) to 100 µl competent cells

17|Incubate the mixture (that is, the cells with DNA) for 1 h on ice.

18|Heat shock cells for at 42°C 1 min.

19|Place the cells for on ice two min.

20|Add 900 µl of LB medium to the cells and incubate at 37°C for 1 h.

21|Centrifuge the cells at 2,600g for 1 min.

22|Remove 900 µl of the supernatant.

23|Resuspend the pelleted cells in the remaining 100 µl supernatant.

24|Plate the resuspended cells onto a LB agar plate with appropriate antibiotics (see supplementary Figure 1 and Table 1).

25|Incubate the plate at 37°C overnight.

The plate can be stored at 4°C.

Supplementary Protocol 2. Overexpression and isolation of GFP-8His

1|Overexpress GFP-8His in BL21(DE3)pLysS harboring pET20bGFP-8His for 4 hours at 25°C essentially as described in Steps 21-26 of the main protocol. Add 0.4 mM IPTG (final concentration) for induction of expression and ampicillin (100 µg/ml) rather than kanamycin.

2|Process cells as described in Steps 22-26 of the main protocol. Remove the supernatant, which contains the GFP-8His, rather than the pellet for use in Step 3.

3|Purify GFP-8His essentially as described in Steps 35-39 of the main protocol. Do not add detergent to Buffers A and B, and wash the Ni-NTA column with 20 column volumes of 10 % Buffer B. Elute GFP-8His with 50 % Buffer B.

4|Pool the fluorescent fractions (GFP-8His-containing) and dialyze overnight in 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8, 100 mM NaCl.

GFP-His8 can be stored at –80°C in small aliquots. Avoid repeated freezing and thawing of GFP-8Hisaliquots.

5|Determine protein concentration with the BCA assay and measure GFP fluorescence, as described in Step9 of the main protocol from 0.01 to 0.3 mg GFP-8His per ml.

Supplementary Protocol 3. Determining amount of membrane protein using GFP fluorescence

1|Plot the GFP fluorescence versus the protein concentration.

2|Use the slope of the plot determined in Step 1 to convert the GFP fluorescence from any 100 l sample to mg of GFP-8His per ml.

3|To account for the dampening of GFP fluorescence in either whole-cells or non-detergent solubilized membrane samples, multiply the values obtained in Step 2 by 1.5 for whole-cells and 1.3 for non-detergent solubilized membranes (see Supplementary figure 2).

4|To estimate the amount of overexpressed membrane protein, divide the molecular weight of the protein by 28 kDa (MW of GFP-8His) and multiply this by the amount of GFP-8His as determined in Steps 2-3.

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