Literature Review

The use of an ultracentrifuge device to pack SSNMR rotors was first mentioned in the literature by Bockmann et al. in 2009.142 The device, used to pack crystalline protein samples into 1.3 - 4 mm rotors using an ultracentrifuge, was a huge development in the packing of such samples. (For further comparison, Das et al. describe a typical home- made device which would have been used to pack these types of samples previously.141) Subsequently, various designs aiming to sediment soluble proteins directly into 1.3 - 4 mm rotors have been published.108, 143–145 Prior to these devices, concentrated samples would have been sedimented into a normal ultracentrifuge tube before being transferred into a rotor, either using a spatula or transferring to a pipette tip and then being centrifuged into the rotor. These multiple steps increase the amount of valuable sample lost and greatly raise the chance of dehydration, emphasising that direct sedimentation

into the rotor is very advantageous. All of the designs presented here for sedimenting protein into a rotor can also be used to pack crystallised proteins.

The packing tool designs currently in the literature are presented in Figure 3.5. Table 3.1 highlights important details of these different ultracentrifuge devices, such as the reservoir volume, which is important when considering sedimentation conditions, and whether they allow for both sedimentation and packing of a sample. It is critical to note that, while these tools have been created for a wide range of SSNMR rotor sizes, currently there are none available for 0.7 and 0.8 mm rotors. Fast MAS (60 - 100 kHz), using 0.7 - 1.3 mm rotors, is often required to sufficiently resolve the complex spectra produced by proteins. Moreover, the sub-milligram amounts of sample required to fill 0.7 and 0.8 mm rotors are advantageous when the samples can be challenging to produce in large quantities and are expensive to isotopically label. For these reasons our ultracentrifuge sedimentation and packing tools are designed for use with 0.7, 0.8 and 1.3 mm rotors.

The packing tool created by Hisao et al. can be used to pack sedimented samples

in addition to crystallised samples, but first requires the sample to be sedimented in a normal ultracentrifuge tube.138 The supernatant is removed before the tube is inverted into the packing tool and the sample is transferred into a rotor using ultracentrifugal force (see Figure 3.5). This has the benefit of a simple sedimentation step where high forces can be used without any concern over damaging the rotor. However, when transferring the sediment to the rotor the chances of protein getting stuck on the walls of the packing device and ultracentrifuge tube are greatly increased, potentially wasting the valuable sample and limiting the amount that reaches the rotor. This highlights the importance of either direct sedimentation into the rotor or minimising the distance that the sediment has to travel to the rotor. The designs by Gardiennet et al.144 and Geliset al.145 both allow for direct sedimentation into the rotor, an improvement on the design by Hisaoet al.

In our design presented below, the sample is sedimented into a narrow reservoir and then directly transferred to a rotor in a second step. The key factor in this decision was to avoid having the rotor present during the sedimentation process, which provides

access to more extreme sedimentation conditions. Thus making it possible for this

packing tool to be used for a greater range of proteins, including those that are typically more challenging to sediment. Although the literature shows that 1.3 mm rotors can survive relatively harsh g-forces,142, 143 it is unknown what forces the more delicate 0.7 and 0.8 mm rotors can withstand.

It is important to note that in the ultracentrifuge device by Bockmannet al.,142 a metal dummy cap was used in place of the bottom rotor cap in order to prevent damage to the delicate rotor caps from the high forces in the ultracentrifuge. This complicates the packing procedure and, more critically, adds a further opportunity for loss or dehydration of the protein. Therefore using a dummy cap should be avoided, if possible, when designing a packing tool.

Figure 3.5: Ultracentrifuge tools in the literature for packing sedimented or crystallised protein into SSNMR rotors: Bockmann et al.,142 Bertini et al.,143 Hisao et al.,138 Gelis et al.,145 _{Gardiennet et al.}144 _{and Mandal et al.}108

Table 3.1: Key details on ultracentrifuge SSNMR rotor packing tools in the literature. (POM is polyoxymethylene, PEEK is polyether ether ketone, CFRP is carbon fibre reinforced polymer and PCTFE is polychlorotrifluoroethylene. SB and FA stand for “swinging bucket” and “fixed angle” ultracentrifuge rotors, respectively.)

Reference Rotors Sediment Conditions Material Volume

-ation?

Bockmann 1.3 – No 210,000 x g (SB) POM, PEEK Unknown

et al. (2009)142 4 mm 30% glass &

epoxide glass

Bertini 1.3, 3.2, Direct 175,000 x g Polycarbonate, 20 or

et al. (2012)143 _{& 4 mm aluminium & 1.38 ml.}

POM

Gardiennet 3.2 mm Direct 210,000 x g CFRP 1 ml

et al. (2012)144

Gelis 3.2 mm Direct 121,000 x g (SB) Aluminium 6 –7 ml

et al. (2013)145 _{201,000 x g (FA) & POM}

Hisao 1.6 & Indirect 3,000 x g PCTFE Unknown

et al. (2016)138 _{3.2 mm}

Mandal 3.2 mm Direct 143,000 x g PEEK 1 – 1.5 ml

The size of the funnel is also an important consideration when designing a sedi- mentation device as this limits the volume of the protein solution and subsequently the total amount of protein in the device. Two different ultracentrifugal devices are pre- sented by Bertiniet al. (labelled as “A” and “B” in Figure 3.5).143 Device A has a very large reservoir (20 ml), which is necessary when sedimenting less concentrated solutions. However, there are downsides to this tool: the polycarbonate funnel in which the solution is placed must be cut to size each time and the tools includes aluminium components, which may corrode. Device B has a much smaller funnel (1.38 ml) and therefore is more suitable for the sedimentation of concentrated samples. It is made from the more chemically resistant polyoxymethylene (POM) and as such can be cleaned with acids or bases if necessary.

Two types of ultracentrifuge rotors are used with these tools: fixed angle and swinging bucket (Figure 3.6). A fixed angle rotor can typically reach higher speeds than a swinging bucket rotor, however the force produced by the rotor will not be parallel to the SSNMR

rotor due to its fixed angle and may even cause damage to the MAS rotor.108 If the

SSNMR rotor is not packed in an axially symmetric fashion, it will not spin stably in the SSNMR probe (Figure 3.7). While the sample is generally viscous and will rearrange itself under MAS, occasionally the critical spinning rate required for this to happen cannot be reached. On the other hand, the swinging bucket rotor, which allows the SSNMR rotor to become parallel to the g-force, is ideal. For the designs presented here, the extreme forces produced by the fixed angle ultracentrifuge rotor (up to 700,000 x g) are initially exploited to sediment the protein sample (SSNMR spectra of proteins sedimented under similar conditions show that the high forces involved do not affect the protein structure).23, 123, 144 The packing device is then transferred to the swinging bucket ultracentrifuge rotor for uniform packing of the sediment into the SSNMR rotor.

The most recent devices designed by Mandal et al. allow sedimentation and

packing into 3.2 and 4 mm rotors.108 They are comprised of 3 pieces each and have

a relatively large volume. However the process of sedimentation into a rotor is quite tedious: it requires three separate hour-long runs (at 154,000 x g) with manual rinsing in between each step. The long packing time is probably due to the limited speed of the swinging bucket rotor. This highlights the advantage of using a fixed angle rotor, which is capable of reaching significantly greater speeds, in a separate packing step. The design presented in this chapter uses forces up to 700,000 x g for sedimentation in comparison to others in the literature which range from 3,000 x g to 210,000 x g.