Materials & Methods

2.1: Materials

2.1.1: Cloning Materials

All enzymes used in the manipulation of DNA were from Boehringer Mannheim unless otherwise stated. Shrimp alkaline phosphatase and Sequenase kits were from USB. All radiochemicals were from Amersham. Taq polymerase was from Cetus, dNTPs and the Ready-To-Go DNA labelling kit were from Pharmacia. Agarose powder was from Flowgen, X-OMAT AR film from Kodak, and Geneclean from BIOIOI. D.discoideum genomic DNA, and DNA from the A.gtl 1 cDNA library was prepared by Molly Strom. General media recipes were prepared according to Sambrook et al (1989). Oligonucleotides were made by Jashu Mistry on an Applied Biosystems 3SOB DNA synthesizer or on a Beckman Oligo 1000 DNA synthesizer. A list o f the oligonucleotides used in this study whose sequences are not given in the results chapters are given in Table 2.1.

Table 2.1 : Oligonucleotides used in this study

Title Sequence Used for

SBL18 5 ’-CCACGTTTGGTGGTGGCGACC ATC-3 ’ _{pGEX-2Tprimer} SBL19 5 ’-CGTCATCACCGAAACGCGCGAGGC-3 ’ _{pGEX-2T primer} SBL45 5 ’-CGCGGATCCGGATTTGATTGTTTTGATATAAAAAAAATG-3 ’ _{PCR cloning DdracGAP into}

pGEX-2T

SBL46 5 ’-GCGGGATCCTTAGGTTGATAAATGATTTGATTG-3 ’ _{PCR cloning DdracGAP into} pGEX-2T

SBL50 5 ’-GCAATCAAATCATTTATCAACATC-3 ’ _{Sequencing DdracGAP gDNA} SBL51 5 ’ -G AACC ATTATTTATTTGG AG-3 ’ _{Sequencing DdracGAP gDNA} SBL52 5 ’-TTTAACCAACCTTCCTCTG-3 ’ _{Sequencing DdracGAP gDNA} SBL53 5 ’ -GTAGAGC AGGGTC ATTCC-3 ’ _{Sequencing DdracGAP gDNA} SBL54 5 ’-TTTTGAAGAGGAGTTGGTGG-3 ’ _{Sequencing DdracGAP gDNA} SBL55 5 ’-TACAAGTGGTGGTGGTGCTG-3 ’ _{Sequencing DdracGAP gDNA} SBL56 5 ’-CCACCAGTTAATAAATCGACG-3 ’ _{Sequencing DdracGAP gDNA} SBL61 5 ’-GTGTTGTTACAATTGTCTCT-3 ’ _{Sequencing DdracGAP gDNA} SBL62 5 ’-GTAGTATAGATAAATAG-3 ’ _{Sequencing DdracGAP gDNA} SBL63 5 ’ - AA AGG ATT AATATTTTTC-3 ’ _{Sequencing DdracGAP gDNA} SBL64 5 ’-TTATTAACTTCTCTCTAC-3 ’ _{Sequencing DdracGAP gDNA}

MS74 5 ’ -GGTGGCGACG ACTCCTGGAGCCCG-3 ’ _A.gtl 1 primer MS75 5 ’ -TTG AC ACC AG ACC A ACTGGTA ATG-3 ’ _{A.gtl 1 primer}

2.1.2: Materials used for protein studies

The cDNA expression constructs pGEX2T-rhoA and pGEX2T-rhoGAP catalytic domain were obtained from Professor Alan Hall (University College, London).

Isopropyl p-D-thiogalacto-pyranoside (IPTG) was from Sigma. Glutathione-agarose and p-aminobenzamidine-agarose were from Sigma. Chromatography columns were purchased from Omnifit. All standard buffer reagents were from BDH. Guanine nucleotides and alkaline phosphatase-agarose were from Sigma, with radiolabelled guanine nucleotides from Amersham International. Nitrocellulose filters used in GDP dissociation assays were from Millipore. Dialysis cassettes were from Pierce. 2 ’(3’)-0- (N-methylanthraniloyl) (mant) derivatives of GTP, GDP, and GMPPNP were made in John Eccleston’s Laboratory by direct acylation of the 2 ’ and 3’ hydroxyl groups of the ribose moiety with N-methylisatoic anhydride (Hiratsuka, 1983; Neal et a l, 1990). The structure of mantGTP is shown in Figure 2.1.

Protein purification was performed using a Waters 650E advanced protein purification system with a Waters 484 tunable absorbance detector and a Gilson 203 fraction collector. HPLC was performed using a Waters HPLC system with a tunable wavelength detector and a Waters injection system, with detection of absorbance changes by a Hewlett Packard integrator. HPLC columns were from Whatman.

2.2 : Protein Methods

2.2.1 : Expression and purification of recombinant proteins from E. coli. All recombinant proteins purified from E.coli in this study have utilised the GST-fusion protein expression system using the plasmid vector pGEX-2T (Pharmacia). For large scale preparations o f recombinant proteins, pGEX-2T constructs in E.coli D H 5a cells were grown in Luria Broth containing 75 pg ml’^ ampicillin at 37^C in flasks on a rotary shaker (New Brunswick Scientific) at 200 r.p.m. Either 6 litre or 40 litre cultures were grown by inoculating pre-warmed media with overnight cultures at a dilution o f 1/50. Cultures were grown up to an optical density o f A6oo=0.8, and were then induced by the

Figure 2.1 : Structure of MantGTP

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addition of IPTG to a final concentration o f 1 mM for 6 litre cultures or 0.1 mM for 40 litre cultures. For recombinant human rhoA and rhoGAP catalytic domain, induction was performed for 3-4 hours at 37^C. For recombinant D. discoideum racl A, rac C, rac 1AQ61l, rac and DdracGAP catalytic domain, induction was performed for 16- 20hours at 22°C, as induction at 37^C gave a lower yield of soluble recombinant protein. After induction cells were pelletted by centrifugation at 3,300 x g for 15 minutes at 4^C. Cells were washed once in an ice-cold buffered solution of 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and then resuspended in this buffer by the addition o f approximately 10 ml o f ice-cold buffer per litre o f original E.coli culture. When 40 litre cultures were grown the cells were immediately lysed using a French Press and then frozen in 40 ml aliquots at -80°C. When 6 litre cultures were grown the cells were frozen at -80^C immediately after resuspension.

To purify recombinant proteins the cell suspension was thawed and, for the 6 litre cultures, cell lysis was acheived by sonication on ice, using a half-inch probe on setting seven (Sonics & Materials Inc., “Vibra Cell”). The 40 litre cultures had been lysed before freezing and simply required thawing, with the equivalent o f 10-15 litres o f original E.coli culture thawed for purification (i.e. 150 ml of frozen lysate). This whole cell extract was ultra-centrifuged at 110,000 x g for 30 minutes at 4°C to pellet the cell debris. The supernatant was applied to a 15 ml Glutathione agarose (Sigma) column (Omnifit) pre­ equilibrated with lysis buffer, and linked to a fraction collector and UV detector, at a flow rate o f 0.6-1 ml m in '\ Protein flowing through the column was monitored at 280nm. After loading, the colunrn was washed with lysis buffer at the same flow rate until the Aisonm o f the eluent had returned to a baseline value. At this point, a 5 ml p- aminobenzamidine (Sigma) column (Onmifit) was linked downstream o f the glutathione agarose column to remove thrombin. The two columns were then equilibrated at the same flow rate with thrombin cleavage buffer : 50 mM Tris-HCl pH7.5, 50 mM NaCl, 10 mM MgCl2, 2.5 mM CaCl2, 1 mM DTT. After equilibration the flow rate was reduced to 80 p.1 m in'’ and a solution of 500 Units o f human thrombin (Sigma) in 50 ml o f thrombin cleavage buffer was applied to the two colunms for 10 hours, with 2.5 ml fractions collected. After application o f the thrombin solution the columns were washed with thrombin cleavage buffer to remove all of the recombinant protein cleaved off the glutathione agarose column. The glutathione agarose column was then washed at 1 ml

min'* with 5 mM glutathione (Sigma) in this buffer to remove the GST protein and any uncleaved GST fusion protein. Finally, both columns were washed with 3 M NaCl to remove any non-specific protein contaminants, and were stored in distilled water with 0.02% sodium azide at 4°C.

The elution profile showing the absorbance at 280 nm o f the eluent from columns during the purification o f recombinant rhoA is shown in Figure 2.2a. This elution profile was similar for all the recombinant proteins purified by this system. Upon application o f the whole cell extract peak A shows the flow-through o f the native E.coli proteins that did not bind to the column. Upon application of the thrombin solution peak B shows the elution of recombinant protein from the column due to thrombin cleavage. Finally, upon application o f 5 mM glutathione, peak C shows the elution of the GST protein and any uncleaved fusion protein. Some of the fractions obtained during purification were analysed by SDS-PAGE and are shown for the purification o f rhoA in Figure 2.2b. It was found that only for the purification of recombinant human rhoGAP did complete thrombin cl!(j‘eavage occur. This is probably because the rhoGAP construct has a longer linker sequence between the GST protein and the rhoGAP protein than do all o f the other constructs. This is because rhoGAP is cloned into the EcoRI site o f pGEX-2T, whereas all other constructs are either cloned into the Smal site (human rhoA) or the BamHI site (all D.discoideum clones).

Cleaved proteins were concentrated to 5-50 mg ml'^ using a Centriprep (Amicon) with a 10 kDa cutoff, by centrifugation at 3,000 x g at 4°C. The concentrated proteins were dialysed (Pierce) against 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 1 mM DTT for small GTPases, and against the above buffer without MgCl2 for GAPs. The dialysed proteins were then dispensed into small aliquots, frozen rapidly in dry ice and stored at -SO^C.

2.2.2: Protein concentration and purity determination 2.2.2.1: BioRad assay

A commercially available dye-binding assay, the BioRad assay (BioRad Laboratories), was used to determine total protein concentrations. Bovine serum albumin (BSA) was used as a standard with a standard curve of BSA concentration versus absorbance at 595nm obtained up to 100 pg BSA added. The absorbance at 595 nm was determined

Figure 2.2 : The purification of recombinant rho A protein from E.coli.

RhoA was purified from E.coli using the pGEX-2T GST fusion protein expression system as described in the text.

(a) Eiution profiie obtained in the purification of rho A

The elution profile showing the absorbance at 280nm o f the eluent from the glutathione- agarose and p-aminobenzamidine columns during the purification of recombinant rhoA is shown. Peak A is the flow-through jfrom the glutathione-agarose column upon application o f the E.coli supernatant. Peak B is the elution of rho A from the column by thrombin cleavage o f rho A from GST. Peak C is the elution o f GST and any uncleaved GST-rhoA from the glutathione agarose column by 5 mM glutathione.

(b) SDS-PAGE anaiysis of fractions obtained in the purification of rho A

Fractions obtained in the purification o f rhoA were analysed by polyacrylamide gel electrophoresis using a 12% gel (Section 2.2.2.3). Lanes 1 and 20 show the molecular weight markers. Lane 2 is the supernatant that was loaded onto the column. Lanes 3-11 are fractions collected during thrombin cleavage. Lane 12 is empty. Lanes 13-16 are fractions collected after thrombin cleavage and during elution with 5 mM glutathione. Lanes 17-19 are purified concentrated rho A protein with loadings o f 1, 2, and 5 pg respectively.

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after 10 minutes and showed a linear dependence on the BSA concentration up to approximately 25 pg m l'\ The concentration of sample protein was determined in the same way as for the BSA standard curve, enabling the calculation o f total protein concentration from the standard curve. This assay was used both for determination o f the protein concentration o f purified recombinant proteins and for total cell extract concentrations.

2.2.2.2: Protein concentration using calcuiated extinction coefficients

The concentration o f highly purified recombinant proteins from E.coli was also determined by ultra-violet spectroscopy using a Beckman DU-70 spectrophotometer and a theoretical molar extinction coefficient. Theoretical molar extinction coefficients were calculated using the molar extinction coefficients of tryptophan, tyrosine, and cysteine residues, and o f GDP (Gill and von Hippel, 1989). The extinction coefficients at 280 nm for the proteins studied in this thesis are shown in Table 2.2. It was found that the concentrations of purified recombinant proteins determined by both BioRad assay and the absorbance at 280 nm agreed to within 10% o f each other.

Table 2.2 : Theoretical extinction coefficients of purified proteins Protein Theoretical extinction

coefficient (cm'^M'^)

Human rhoA 25,900

Human rhoGAP catalytic domain 13,370

D.discoideum racl A 30,128 D.discoideum raclA^^^^ 30,128 D.discoideum rac C 30,008 D.discoideum racC^^"^^ 30,008 D.discoideum DdracGAP 18,020

2.2.2.3: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-polyacrylamide gel electrophoresis was used to determine the purity o f proteins throughout purification procedures. A BRL gel apparatus was used with gels o f 180 x 120 X 0.8 mm. The stacking gel consisted of 4% polyacrylamide, 130 mM Tris-HCl pH6.8, 0.1% SDS, and the resolving gel contained 12% polyacrylamide, 390 mM Tris-HCl

pH 8.8, 0.1% SDS. Protein samples were heated to lOO^C in 1 x Laemmli buffer (Sambrook et a l, 1989), with typically a volume o f 20 pi sample loaded per lane. Gels were electrophoresed at 60 mA until the bromophenol blue dye front was run off the gel in a running buffer o f 25 mM Tris, 250 mM glycine pH8.3, 0.1% SDS. Proteins were stained with 2 gL‘' Coomassie brilliant blue R250 (Sigma) and 0.5gL’' Coomassie brilliant blue G250 (Sigma) in 45% ethanol, 10% acetic acid, and 5% methanol. Gels were stained for one hour and then destained in 10% acetic acid, 10% methanol.

2.2.3: HPLC analysis of guanine nucieotides

Guanine nucleotides and mant guanine nucleotide analogues were separated on a I

Whatman Partis^l SAXIO column (0.46 x 25 cm). The column was eluted isocratically at 1.5 ml m i n ’ using 0.6 M monobasic ammonium phosphate buffer (pH4.0) for native guanine nucleotides, and supplementing this buffer with 25% methanol for mant guanine nucleotide analogues. For native guanine nucleotides, the eluent was monitored for nucleotide absorbance at 252 nm using a Waters lambdamax 481 LC spectrophotometer. For mant guanine nucleotide analogues the eluent was monitored for mant fluorescence using a Hitachi F-1050 fluorescence spectrophotometer, exciting at 366 nm and measuring the emission at 440 nm. For both native guanine nucleotides and mant guanine nucleotide analogues, the areas of the eluent peaks obtained were calculated by an HP 3390 integrator. Typical nucleotide loading amounts were 2 nmoles for native guanine nucleotides and 75 pmoles for mant guanine nucleotide analogues in a volume o f 100 pi. The injection of guanine nucleotides and mant guanine nucleotide analogues onto the

I

Whatman Partis^l SAXIO column hoth separately and as mixtures enabled the determination o f the retention time for each nucleotide species. These retention times were : GDP = -4.6 minutes, GTP = -12.8 minutes, GMPPNP = -9.8 minutes, mantGDP = -3.7 minutes, mantGTP - 8 minutes.

2.2.4: Formation of complexes of rhoA with mant guanine nucleotide analogues and GTP

Complexes between purified rhoA and mantGTP, mantGDP, mantGMPPNP and GTP were prepared by fast exchange o f the nucleotide into the protein. A twenty-fold excess o f the nucleotide was added to 50 nmoles of rhoA in a buffer of 50 mM Tris-HCl pH7.5, 40mM EDTA, 1 mM DTT in a total volume of 450 pi at room temperature for 10

minutes. The reaction was quenched by the addition o f MgCl2 to a final concentration of lOOmM (50|o,l o f 1 M MgCl2 added), and the rhoA.nucleotide complex separated from free nucleotide by gel filtration on a PD-10 column (Pharmacia) at 4^C. The PD-10 column was pre-equilibrated with a buffer of either 50 mM Tris-HCl pH7.5, 2 mM MgCl2, 1 mM DTT for the experiments in Section 3.2, or 20 mM Tris-HCl pH7.5, 2 mM MgCl2, 1 mM DTT for the experiments described in Section 3.3/4. The passage o f the complex (followed by free nucleotide) was monitored for mant nucleotide exchanges using an ultra-violet light source, and for complexes with GTP by collection o f 300 pi fractions followed by determination of the A280nm- The concentration o f mant complexes was determined using the extinction coefficient o f mant at 350 nm (5700 M'^cm’^), and the theoretical extinction coefficient o f rhoA.mant nucleotide at 280 nm (26,959 M’^cm’^). The concentration o f rhoA.GTP complexes was determined using the theoretical extinction

separation.

extinction coefficient o f 25,900 M*' c m '\ Typically, 80% recovery was acheived after

2.2.5: Formation of complexes of rhoA with GMPPNP

Complexes between purified rhoA and GMPPNP were prepared in a similar manner to that described by John et al (1990). The principle of the exchange reaction is to incubate rhoA.GDP with GMPPNP in the presence of alkaline phosphatase. The alkaline phosphatase only hydrolyses GDP and GTP and not GMPPNP under the conditions used. Exchange conditions were 200 pM rhoA.GDP, 400 pM GMPPNP, 20 mM Tris-HCl pH 7.5, 25 mM NaCl, 4 mM MgCl2, 1 mM DTT, 200 mM (NH4)2S04, 10 U ml'^ alkaline phosphatase agarose, in a total volume o f 1 ml. Exchange was performed for 90 minutes at room temperature with gentle agitation after which the alkaline phosphatase agarose was removed by centrifugation at 16,000 x g for 2 minutes. The protein was dialysed against a buffer o f 20 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT, concentrated to 200 pM, and the identity o f the bound nucleotide confirmed as 100% GMPPNP by HPLC analysis as described in Section 2.2.3.

2.2.6: Stopped-flow fluorescence measurements

All stopped-flow experiments used a Hi-Tech Scientific SF-61MX spectrofluorimeter. Monochromatic light at 366 nm was obtained from a lOOW mercury arc lamp using a monochromator. Emitted light from the 2 mm by 2 mm path length cell was measured

through a Wratten WG 47B bandpass filter. Data was collected and analysed using RK2 software (Hi Tech Scientific). Analysis of data was also performed using Excel and Grafit programs. The data were collected using both linear and logarithmic timescales, where use o f a logarithmic timescale resulted in greater data points at short timepoints and fewer data points at longer time points. The use o f a linear or logarithmic timescale is indicated in the legends to specific figures. The spectrofluorimeter was used in both the L- and T- formats, where the L-format involved the use of a single photomultiplier tube for measurements of fluorescence intensity. The T-format involved the use of two photomultiplier tubes with polarising filters so that both parallel and perpendicular fluorescence could be measured. This enabled not only the determination o f fluorescence intensity as intensity = In+2Ix, but also the determination o f fluorescence anisotropy as anisotropy = (IirIi)/(In+2I J .

All concentrations of reagents quoted, refer to final concentrations after mixing rather than to syringe concentrations. Multiple traces were obtained for each reaction which were then averaged and fitted to appropriate equations. Therefore all error bars represent standard errors of the data fi*om the average rates.

2.2.7: Quenched-flow measurements

Quenched-flow experiments used a Hi-Tech micro volume rapid quench flow system. The system contains three syringes, two for the reagents and one for the quenching solution. Equal volumes of the reagents and quench are used (150 pi o f each). Different timed solutions up to 167 msec were obtained by the concomitant use of different loop lengths and flow rates before quenching. Longer time points (>167 msec) were obtained by using a time-delay mode where the resultant mixture remained in a loop before quenching. Reaction mixtures were displaced from the collection loop with 500 pi water and were retrieved from the collection valve. All reagent concentrations refer to final concentrations after mixing unless otherwise stated.

2.2.8: Slow time-course fluorescence measurements

All slow time course fluorescence measurements were performed on an SLM 8000S spectrofluorimeter, in the L-format, using 10 mm by 10 mm quartz cuvettes. Excitation of the mant moiety was at 360 nm with the emission wavelength stated for each experiment

along with the excitation and emission slit widths. All data were collected manually with the shutters closed between readings to prevent photobleaching, and a control cuvette included to normalise for fluctuations in lamp intensity and/or photomultiplier response.

2.2.9: The GDP dissociation assay

In this assay the protein o f interest was initially exchanged with [8-^H]GDP by incubating 115 pM protein with 41.5 pM GDP (8.5 pM [8-^H]GDP and 33 pM GDP) in a buffer of