Chapter Common materials and methods 2.1 DNA cloning 2.1.1 Sequence information The hNck2 protein sequence was obtained from the National Center for Biotechnology Information (NCBI). A DNA sequence encoding hNck2 was designed by choosing E.coli-preferred codons. Cdc42 and IRSp58 constructs were obtained from Sohail Ahmed’s laboratory. Others DNA fragments were either obtained by PCR-based de novo design or by RT-PCR. 2.1.2 Bacteria strains and plasmids The bacterial strains are E.coli strains: DH5α and BL21. The plasmids used in this thesis are: pET32a (Novagen), pGEX4T-1(Promega) and pGEX6P-1(Amersham). E.coli strains were grown on LB agar plates or LB broth with ampicillin (Amp, 100μg/ml). Stock cultures of E.coli were maintained at -80℃ as a suspension in a supplemented LB broth containing 10%( v/v) glycerol and 50mM CaCl2. 2.1.3 Plasmid extraction E.coli strains were grown in LB broth (Bio Basic INC.) cultured at 37℃ overnight. Plasmids were extracted using QIAprep Spin Miniprep Kit 250, according to the manufacturer’s protocols. After the mini-preparation, the purified plasmid was dissolved in the TE buffer (10mM Tris-HCl and 1mM EDTA, pH 7.5) and stored at -20℃ for future use. 45 2.1.4 Cloning DNA fragments and transformation into E.coli strains PCR amplified DNA fragments or DNA fragments obtained by PCR-based de novo gene synthesis were further digested by BamH1 (N-terminal, New England Biolab) and Xho1 (C-terminal, New England Biolab) at room temperature for three hours. After DNA purification (QIAquick Gel Extraction Kit), a ligation reaction was conducted at 4℃ for 48 hours in the presence of T4 DNA Ligase (New England Biolab). All of the procedures were monitored by Agarose DNA electrophoresis (BioRad). The E.coli DH5α competent cells were prepared and a Heat-Shock transformation was carried out according to the standard protocol. After transformation, the bacteria were recovered in an LB broth and plated on an LB agar (Bio Basic INC.) plate containing ampicillin. 2.1.5 Analysis of plasmid DNA To check the potential clones with target DNA fragments inserted in plasmids, a double digestion was carried out in the presence of BamH1 and Xho1 restriction enzymes. Briefly, 5ml of overnight bacterial culture was obtained and spun at 3000 rpm for five minutes. Plasmids were extracted using QIAGEN Plasmid Mini Purification Kit according to the manufacture’s instructions. The raw digested DNA samples were analysed by gel electrophoresis, using 1%(w/v) agarose gel (Ultra PureTM Agarose, Invitrogen) followed by staining ethidium bromide. Clones containing the right insertion were chosen for further experiments. 2.1.6 DNA sequencing The ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Ready Reaction Kit was used to perform a PCR reaction sequencing (Applied Biosystems). The 46 sequencing reaction mixture consisted of 4.2 μl of autoclaved water, about 300 ng of the plasmid DNA, 0.8 μl of 2.5 μM primer and μl of BigDye. The PCR reaction sequencing was carried out in an Eppendof Mastercycler Gradient PCR machine using the following programme: One minute 96 ℃, 27 cycles of denaturation 96℃, 10s, annealing 50℃, 5s, extension 60℃, one minute, 45 seconds. The PCR product was precipitated by NaAc solution (3 μl of NaAc (pH 3.6), 12.5 μl ddH2O, 62.5 μl 100% ethanol). After being kept at RT for 15 minutes and put through a subsequent centrifugation at 13000 rpm for 20 minutes, the pellet obtained was washed twice with 500 μl of 70% ethanol. Later, the pellet was air dried and stored at -20℃. Prior to sequencing, 12 μl of Hi-DiTM formamide (Applied Biosystems) was added to dissolve the pellet and the sample mixture was loaded into a 96-well sequencing plate. DNA sequencing was carried out on ABI PRISM® 3100 genetic analyser, using the dye termination method. The sequence was analysed by DNAman or Vector NTI (InforMax). 2.2 Protein expression and purification 2.2.1 Unlabelled protein expression and purification The transformed E.coli BL21(DE3) cells were grown overnight at 37℃ to maximal density in 5ml of an LB medium, containing 100μl/ml ampicillin and then diluted to L of an LB medium, containing 100μl/ml ampicillin. Cells were grown at 37℃ until OD600 reached 0.6, and subsequently induced with 1.0 mM IPTG (Invitrogen). After further incubation at 37℃ for 3.5 hours or 20℃ for 16 hours, the cells were harvested by centrifugation (6000 rpm) for 30 minutes at 4℃(Beckman). 47 The cell pellet was resuspended in 40ml ice-colded phosphate buffer (PBS; 50mM NaH2PO4 (Kanto Chemical Co. Inc.), 150mM NaCl (Merch), pH 7.4) and sonicated on ice (Branson Sonic Power Co.). The cell lysate was isolated by centrifugation (18000 rpm) for 30 minutes at 4℃. The supernatant was loaded on 1ml GlutathioneSepharose 4B resin (Amersham), which was pre-equilibrated with PBS, or on 1ml of Ni-NTA magnetic beads (Amersham), which were equilibrated with PBS (depending on fusion types), and incubated at 4℃ for one hour. The resin was subsequently washed three times with 10 column volumes of PBS each time. With the addition of 40 units of Thromin (Sigma) per ml of resin, the proteins were released from GST after four hours of incubation at room temperature; his-tag fusion proteins were directly eluted by adding a 250mM imidazole solution buffer. Under denatured conditions, the pellets were resuspended in a 40ml 1% triton 100 solution buffer, and subsequently isolated by centrifugation (18000 rpm) for 30 minutes at 4℃. The pellets were again resuspended in an 8M urea buffer, and isolated by centrifugation under the same conditions. The supernatant was loaded onto 1ml Ni-NTA magnetic beads, which were pre-equilibrated in an 8M buffer, and incubated for one hour at 4℃. The denatured his-tag-fusion proteins were eluted from the resin by adding 250mM of imidazole. 2.2.2 Isotope labelled protein expression and purification 15 N-labelled proteins and 15 N, 13C-labelled proteins were prepared, following similar expression and purification procedures, except for growing E.coli cells in the M9 minimal medium (Na2HPO4·12H2O 17.1g/L(Kanto Chemical Co., Inc); KH2PO4 3g/L(Kanto Chemical Co., Inc); NaCl 0.5 g/L(Merck KGnA); (15NH4)2SO4 48 1g/L(Cambridge Isotope Laboratories Inc); Glucose 4g/L (aMReSCo) or 13 C6H12O6 2g/L (Cambridge Isotope Laboratories Inc); MgSO4 mM (Merck); Thiamine(VB1) 2mg/L (Sigma); ampicillin 75mg/L) instead of an LB medium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, using a 15% (w/v) polyacrylamide gel to check protein expression and purification (Bio-Rad). 2.2.3 Further purification by HPLC and FPLC For further purification, reverse-phase HPLC was performed by using a 218TP510 C18 or 208TP510 C8 semi-preparative reverse-phase column (Vydac). 10% acetic acid was added in protein solution and it was filtered before injection. Different fractions were separated and eluted with a gradient of 0-50% (v/v) acetonitrile (Merck), containing 0.1% trifluroacetic acid (TFA, Fisher Scientific) at a flow rate of 6.0ml/min. The elution was monitored at both 215nm and 280nm. All the peaks with an absorbance over 0.5 at 215nm were collected and lyophilised. The molecular weight of these peaks was confirmed by MALDI-TOF MS. Alternatively, ion-exchange chromatography and gel filtration was performed on a Mono-S HR 5/5 column or a Sephadex G-200 column, respectively, in different cases. The elution of the protein was pre-equilibrated with a running buffer. Ion-exchange chromatography was performed with a gradient 0-1M NaCl buffer at a flow rate of 1ml/min. Gel filtration was performed at a flow rate of 1ml/min. The fractions were auto-collected for molecular weight determination and further buffer exchange. 2.2.4 Determination of Protein Concentration 49 The concentration of a protein solution is usually determined by measuring the absorbance at 280nm and using the Bear-Lambert Law: A = ε*l*C, where ε is the molar absorption coefficient (M-1cm-1), l is the path length (cm), and C is the protein concentration (M). For proteins containing Trp residues, it was recommended that the ε could be best estimated by the following equation: Ε(280)(M-1cm-1)=(#Trp)(5,500)+(#Tyr)(1,490)+(#Cystine)125 Here, #Trp, #Tyr and #Cys are the numbers of each corresponding residue in a protein. (Pace et al., 1995) For proteins/peptides without Trp or Tyr, a BCATM Protein Assay Kit was used to determine the protein concentration (Wiiechelman, K, 1988). Procedures were carried out according to the standard BCA protocol. 2.3 CD characterisation of protein structures All CD measurements were carried out on a Jasco J-810 spectropolarimeter, equipped with a thermal controller and using a 1mm pathlength cuvette. Proteins were dissolved in a 5mM phosphoate buffer, with a final concentration around 5μM. FarUV CD spectra were acquired in the range of 190–260nm, while the near-UV CD spectra were acquired in the range of 250–360nm with three independent scans. Thermal unfolding experiments were acquired from ℃ to 95℃ at intervals of five degrees by monitoring ellipticity at 220 nm. 2.4 ITC (Isothermal Titration Calorimetry experiments) characterisation of binding After centrifugation for 15 minutes, the samples were degassed for 15 minutes, to prevent the formation of bubbles. The protein samples were then placed in the 50 reaction cell, and ligands were loaded into the 300 μl injection syringe. Normally, the sample concentration in a syringe is 20-fold of the one in the cell. Titrations were performed at 30℃, and control experiments (peptides titrated into a buffer alone) were conducted to evaluate the heat of dilution. The titration data, after subtracting the corresponding blank results, were fitted, using the built-in software ORIGIN to obtain thermodynamic parameters. 2.5 NMR Experiments and Structure Determination The NMR samples were prepared by dissolving the lyophilised proteins into 0.4 ml of a 20 mM phosphate buffer, or by buffer exchange with a 20 mM phosphate buffer. About 10 % of D2O(Sigma) was added to provide a deuterium lock signal for the NMR spectrometers. In addition, the cocktail of protease inhibitors (Sigma) was also added to inhibit the activities of any residual proteases. NMR experiments included H NOESY, TOCSY, 1H-15N HSQC, 1H-15N HSQC-TOCSY, 1H-15N HSQC-NOESY, HNCACB, CBCA(CO)NH, HCCH-TOCSY, HCCH-NOESY, HNCO. For relaxation experiments, T1, T2 and Heteronulear NOE were also carried out. Spectra processing and analysis were carried out using the NMRpipe (Bruker) and NMRview software (Johnson et al., 1994) Sequence-specific assignments were achieved through connectivities between the pair of HNCACB and CBCA(CO)NH. In structure calculations, NOE peaks were categorised into three groups, based on their intensities: strong, medium and weak, corresponding to the upper bound interproton distances of 3.0, 4.0 and 5.0Å, respectively. The sum of the van der Waals radii of 1.8 Å was set to be the lower bound distance. Backbone dihedral angle restraints were derived from TALOS 51 prediction results, based on Hα chemical shifts (Cornilescu et al., 1999). Hydrogen bond restraints obtained from an H2O/D2O exchange experiment, dihedral angle and manually assigned NOE were included in the calculations, using Cyana2.1 software (Guntert, P., 2004). The final unambiguous NOE assignment combined with other restraints were utilised for structure calculation with distance geometry/simulated annealing protocol implemented on CNS (Brunger et al., 1998). The accepted structures were checked by a PROCHECK (Laskowski R A, et al., 1993) program and analysed by MolMol graphic software (Koradi et al., 1996). 15 N T1 and T2 relaxation times and {1H}-15N steady-state NOEs of the wild type SH3- and SH3-3 were determined on the 800 MHz spectrometer at 20 °C. 15N T1 values were measured from the HSQC spectra acquired with relaxation delays of 10, 500, 100, 600, 200, 300, 400, 900 and 700 ms. 15 N T2 values were determined with relaxation delays of 10, 60, 30, 100, 80, 120, 160 and 180 ms. {1H}-15N steady-state NOEs were obtained by acquiring spectra with and without 1H presaturation, with a duration of s, plus a relaxation delay of s at 800 MHz. 15 N T1 and T2 relaxation times and {1H}-15N steady-state NOEs of the wild type SH3-1 were determined at °C under pH 2.0. 15 N T1 values were measured from HSQC spectra acquired with relaxation delays of 10, 500, 50, 300, 100, 400 and 200ms. 15 N T2 values were determined with relaxation delays of 10, 50, 80, 120, 150, 180, 210, 230 and 250 ms. 15 N T1 and T2 relaxation times and {1H}-15N steady-state NOEs of the wild type SH3- 1(pH 6.5) were determined at °C. 15N T1 values were measured from HSQC spectra acquired with relaxation delays of 10, 400, 100, 300, 200, 350 and 250 ms. 15 N T2 values were determined with relaxation delays of 10, 30, 45, 60, 75, 90 and 150ms. 15 N T1 and T2 relaxation times and {1H}-15N steady-state NOEs of SH3-1-V22 52 (insertion of Val at 22nd) were determined at 20 °C. 15 N T1 values were measured from HSQC spectra acquired with relaxation delays of 10, 700, 100, 600, 200, 500, 300 and 400 ms. 15N T2 values were determined with relaxation delays of 10, 60, 100, 130, 160, 200, 230 and 260 ms. 2.6 Reduced spectral density mapping Calculations of the reduced spectral density mapping need only the R1, R2 and NOE at one field. This is only a simple calculation using the following equations: J(0) = -1.5 / (3.0*d + c) * (0.5*r1 - r2 + 0.6*sigma_noe) J(X) = 1.0 / (3.0*d + c) * (r1 - 1.4*sigma_noe) J(H) = sigma_noe / (5.0*d) where J(0), J(X) and J(N) are, respectively, the spectral density at the zero frequency, at the nitrogen frequency and at the apparent proton frequency (sometimes called J(H) or J(0.87H) ). The other constants and variables are: c = CSA constant d = dipolar constant sigma_noe = cross-relaxation rate (calculated using NOE and R1) 53 . delays of 10, 500, 50, 300, 100, 400 and 20 0ms. 15 N T 2 values were determined with relaxation delays of 10, 50, 80, 120 , 150, 180, 21 0, 23 0 and 25 0 ms. 15 N T 1 and T 2 relaxation times and. (Applied Biosystems). The 47 sequencing reaction mixture consisted of 4 .2 μl of autoclaved water, about 300 ng of the plasmid DNA, 0.8 μl of 2. 5 μM primer and 2 μl of BigDye. The PCR reaction. 1993) program and analysed by MolMol graphic software (Koradi et al., 1996). 15 N T 1 and T 2 relaxation times and { 1 H}- 15 N steady-state NOEs of the wild type SH3- 2 and SH3- 3 were determined