Experimental Details

3.8.1 Information on the Ultracentrifuge

The ultracentrifuge used with these tools was the Beckman Coulter Optima MAX-XP ultracentrifuge fitted with either the MLA-150 fixed angle rotor (for sedimentation) or the MLS-50 swinging bucket rotor (for packing). The internal dimensions of the MLA- 150 and the MLS-50 are different, so each requires a packing tool of different dimensions. The smaller MLA-150 rotor is used for the sedimentation step, due to the higher forces

attainable. For packing the sediment, this smaller packing tool is placed inside an

adaptor so that it fits into the larger MLS-50 rotor (see Figure 3.10). The maximum speeds and forces achievable with the different ultracentrifuge rotors are reported in Table 3.5.

3.8.2 Dimensions and Materials of Tools

All parts of the tools were made at the University of Warwick in the Department of

Chemistry’s mechanical workshop. AutoCAD 2017 was used to produce technical draw-

ings of the parts.

The MLS-50 tool (for packing samples) has 3 components; a funnel, a holder and a rotor sleeve, which fits either a 0.7, 0.8 or 1.3 mm rotor inside. The MLA-150 tool (for sedimentation) has 4 components; a funnel, a holder, a rubber seal and a plug. All components, except the O-ring and rubber seal, are made out of PEEK. The rubber seal is made ofPolyco chemprotect rubber. Technical drawings of all components of the rotor packing tools are provided in Figure 3.22.

3.8.3 Testing of the Tools

Each set of tools was tested as thoroughly as possible in each mode (both sedimentation and packing for each rotor size). Typically, this involved initial tests with water to check the basic functioning of the tool and to test for leaks, followed by using “test” protein samples of either lysozyme crystals for packing or bovine haemoglobin solutions for sedimentation. These proteins were selected due to their low cost, availability and relative simplicity to crystallise or sediment. Finally, once suitable protein samples were ready to be packed into these small rotors for SSNMR, the tools were used to pack these “real” samples.

The maximum forces used in these tests are reported in Tables 3.6 and 3.7, alongside the maximum time the tools were used under these conditions in a single run. Table 3.5: Maximum speeds of the MLS-50 and MLA-150 ultracentrifuge rotors and the calculated maximum force in each case.

Rotor Maximum Speed Maximum Force

MLS-50 50,000 rpm 258,000 x g

Figure 3.22: Technical drawing of the swinging bucket MLS-50 tool (for packing and sedimentation), fixed angle MLA-150 tool (for sedimentation) and the adaptor. All lengths are in mm. “R” preceding a value denotes the radius of a curve.

The cumulative time that each set of tools has been tested at this force is obviously much longer than stated in the tables, for example the MLA-150 tool in sedimentation mode has been tested at 700,000 x g for over 280 hours in total. Note that it will not always be necessary to use forces as high and times as long as those stated here.

3.8.4 Protein Samples

The [U-1H,13C,15N]GB1 sample was prepared, as described previously,13 by expressing the proteins in minimal media with isotopically-labelled with [U-13C]glycerol and [U-

15_{N]-ammonium chloride as the sole sources of carbon and nitrogen. In order to test}

sedimentation in the tools, 25 mg/ml and 50 mg/ml solutions of bovine haemoglobin in sodium phosphate buffer (pH 5.5m, 50 mM) were produced.

Crystallisation

400µl sodium phosphate buffer (pH 5.5m, 50 mM) was added to 4.0 mg of [U-1H,13C,15N]GB1, causing the protein to dissolve. 3 x 400 µl aliquots of the precipitant (2:1 2-methyl- 2,4-pentanediol (MPD) : isopropanol) were added to the protein solution causing it to become cloudy. The solution was thoroughly mixed and stored in the fridge for at least 48 hours to allow the protein to crystallise.

DSS (4,4-dimethyl-4-silapentane-1-sulphonic acid) was added to the crystallised protein sample as an internal reference. 3.0 mg of [U-1H,13C,15N]GB1 crystals were packed into a 1.3 mm rotor, and 0.5 mg into a 0.7 mm rotor.

3.8.5 NMR Experiment(s)

The NMR experiment was recorded on a Bruker Avance III HD spectrometer, using the software Topspin 3.5pl7, operating at 16.4 T (ω0H/2π= 700 MHz) with a Bruker 0.7 mm triple-resonance probe. The measurements were performed at 100 kHz spinning frequency at a sample temperature of 13 ±1°C. The specified sample temperature was achieved by cooling with air (flow of 500 L/h with a target temperature of 15°C) using a Bruker BCU-X cooling unit.

The sample temperature (°C) was measured by the 1H chemical shift of water

with respect to DSS (calibrated to 0 ppm):

T emperature= 568.8−115.8×(δH2O−0.002×(pH−7.4)−0.009×(salt/100)) (3.6)

WhereδH2Ois the chemical shift of water andsaltis the salt concentration in mM.152, 153

Table 3.6: The maximum forces used when testing theMLA-150packing tool for each

mode. The length of time under these conditions in each case is also reported.

Mode of MLA-150 Tool Maximum Force Time at this Force

Sedimentation 700,000 x g 64 hours

Packing into 0.7 mm 20,000 x g 20 minutes

Packing into 0.8 mm 20,000 x g 15 minutes

Table 3.7: The maximum forces used when testing the MLS-50 packing tool for each mode. The length of time under these conditions in each case is also reported.

Mode of MLS-50 Tool Maximum Force Time at this Force

Sedimentation 200,000 x g 90 hours

Packing into 0.7 mm 60,000 x g 10 minutes

Packing into 0.8 mm 60,000 x g 10 minutes

Packing into 1.3 mm 100,000 x g 10 minutes

The measurement was performed using a1H-detected13C-1H 2D pulse sequence. The1H and13Cπ/2 pulses were of 150 kHz and 83.3 kHz nutation frequency, respectively. Adiabatic double quantum cross-polarization154from1H to13C and back to1H was used (contact times 0.2 ms and 0.2 ms,ω1H/2π≈20 andω13C/2π≈80 kHz). 1H WALTZ6449 (ω13C/2π = 10 kHz) decoupling was applied during t1 evolution (acquisition time of 12 ms). 13C WALTZ64 (ω13C/2π = 10 kHz) decoupling was applied during t2 acquisition (acquisition time of 30 ms). MISSISSIPPI51 solvent suppression was used with 70 ms of 50 kHz saturation1H field on resonance with the solvent. The spectrum in Figure 3.18 was the result of 8 scans and had an experimental time of 40 minutes.