Purification and preparation of spin labelled vimentin

Reagents and equipment

FPLC, e.g. Pharmacia

Source S column

Spin label compound (0-87500, (1-oxyl-2,2,5,5,-tetramethyl-A3-pyrroline-3-

methyl) methanethiosulfonate [MTSL], Toronto Research Chemicals, Toronto, Canada)

Superose column

TCEP (tris-(2-carboxylethyl) phosphine, Molecular Probes, Eugene, OR)

Procedure

1. Dissolve IBs (from 500 ml of bacterial culture) in 8 ml of 8 M urea (20 mM Tris pH 8, 1 mM EDTA, 8 M urea) and filter through a 0.2 micron filter (Pall Serum Acrodisc, Fisher Scientific).

2. Chromatograph 4 ml of inclusion body solution on a Hi Load 16/60 Superdex 200 column. The column is run with 20% buffer B, giving conditions of 20 mM Tris, pH 8, 1 mM EDTA, 0.2 M NaCl. Electrophorese column fractions on an SDS-PAG and visualize the proteins by Coomassie blue staining. Pool peak fractions.

3. Desalt the pooled vimentin peak by chromatography over a High-Prep26/10 desalting column.

4. Concentrate the desalted vimentin by chromatography over a Source 15 S column. The peak, typically 2.0 ml, is used for spin labelling.

5. Add TCEP (100 mM stock in H2O) to a final concentration of 100 |xM and incubate at room temperature for 30 min.

6. Spin label the reduced vimentin by addition of spin label to a final concentration of 500 |xM; continue incubation for 1 h.

7. After spin labelling, add 8 ml of buffer A (8 M urea, 20 mM tris, 1 mM EDTA, pH 8.0) and chromatograph the spin labelled protein over the Source 15S as before. Collect the purified peak and store at -800C.

1 2345678M

Figure 6.36 Representative spin-labelled vimentin samples. Samples of Source S fractions 21 and 22 of spin-labelled vimentin mutants cys342, cys345, cys346 and cys349 are shown following electrophoresis on SDS-PAGE and staining with Coomassie Blue. In each pair of lanes, fraction 21 is first, followed by fraction 22; Lanes 1, 2: vimentin cys342; lanes 3,4, vimentin cys345; lanes 5,6, vimentin cys346; lanes 7,8, vimentin cys349. Markers in lane M are Benchmark protein standards from Invitrogen. Vimentin migrates between the 50 and 60 kD bands

Figure 6.36 shows the results of the above purification/labelling scheme, for four separate vimentin mutants.

D. Assembly of intermediate filaments

Equipment

Dialysis tubing and clips, e.g. Spectra/Por 6 regenerated cellulose, 10000 molecular weight cut-off dialysis tubing (Fisher Scientific)

Procedure

1. Assemble purified vimentin in 8 M urea into filaments by dialysis against buffers without urea. Single-step dialysis can be performed using 20 mM Tris pH 7.5 and either 160 mM KCl or NaCl [6,7]. Dialysis is performed at room temperature, overnight. For EPR studies, protein concentrations > 1 mg/ml are used. For observation of filaments by electron microscopy, protein concentrations

Nuclear Matrix
Figure 6.37 Intermediate filaments assembled from spin-labelled vimentin 342C. Spin-labelled vimentin was assembled by dialysis against 20 mM Tris pH 7.5, 160 mM NaCl, overnight followed by negative staining and visualization by EM

of 0.2-0.5 mg/ml are used. Figure 6.37 shows an example of filaments assembled from vimentin cys342.

2. If EPR spectra are to be recorded at multiple steps during vimentin assembly, dialysis can be performed in a stepwise fashion [8]. Starting conditions are 20 mM Tris pH 8.0, 1 mM EDTA, 8 M urea (buffer A + 8 M urea). Vimentin dimers can be produced by dialysis against buffer A + 4 M urea. Vimentin tetramers can be produced by dialysis against 5 mM Tris pH 8 [9]. Filaments can be assembled from any of these intermediate steps by dialysis against assembly buffer, 20 mM Tris pH 7.5, 160 mM NaCl. Perform dialysis for 6 -8 h, followed by overnight dialysis, for filament formation.

EPR measurements can be carried out in a JEOL X-band spectrometer fitted with a loop-gap resonator (Molecular Specialties, Inc., Milwaukee, WI), or equivalent [10,11]. An aliquot of purified, spinlabelled protein is placed in a sealed quartz capillary (0.84 mm OD, Ruska Instrument

8 M urea

4 M urea

Figure 6.38 Normalized EPR spectra from spin-labelled vimentin cys342. Spectra were collected from monomers (8 M urea), dimers (4 M urea) and filaments (20 mM Tris pH 7.5, 160 mM NaCl)

8 M urea

4 M urea

Figure 6.38 Normalized EPR spectra from spin-labelled vimentin cys342. Spectra were collected from monomers (8 M urea), dimers (4 M urea) and filaments (20 mM Tris pH 7.5, 160 mM NaCl)

Corp., Houston, TX) and inserted into the resonator. Spectra of samples at room temperature (20-220C) were obtained by a single 60 s scan over 100 G at a microwave power of 2 mW, a receiver gain of 250-400 and a modulation amplitude optimized to the natural line width of the individual spectrum. For intact filaments, spin label concentration is typically in the range of 50-70 ^M. Intermediate filament samples tend to aggregate upon further concentration, so instrument sensitivity should be optimized to obtain good signal to noise, especially for specimens displaying broad line widths. However, protofilaments of IF oligomers formed in low ionic strength, can be concentrated using centrifugal devices providing excellent signal to noise ratio with even less sensitive instrumentation. For spectra obtained at -1000 C, the microwave power is reduced (100 |xW) to avoid saturation. A representative example of EPR data is shown in Figure 6.38.

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