4. Outlook 76
5.1.6. Plasmids
Table 5.3.: Plasmids used and constructed in this study
Name Description Source
pRosetta rare tRNAs (cmR) lab collection
pSUMO pET24 based with N-terminal His6-Smt3-Ulp1 site(SUMO)-tag,T7 pro-motor, kanR
Andreasson et al.
2008b
pmCUP313 ampR, HIS3, CUP1 lab collection
pSUMOZuo His6-SUMO-Zuo, kanR Koplin et al. (2010) pSUMOSsz His6-SUMO-Ssz, kanR Koplin et al. (2010) pSUMOSsz,Zuo His6-SUMO-Ssz-Zuo, kanR Koplin et al. (2010) pSUMOZuo∆N62 His6-SUMO-Zuo ∆N62, kanR this study
pSUMOZuoH128C/
C167S
His6-SUMO-Zuo H128C C167S, kanR
this study pmCUPZuoC167S pmCUP313-Zuo C167S, ampR this study pmCUPZuo∆N62 pmCUP313-Zuo∆N62, ampR this study
pmCUPZuo pmCUP313-Zuo, ampR this study
pmCUPZuoE30R pmCUP313-Zuo E30R, ampR this study pmCUPZuoP31A pmCUP313-Zuo P31A, ampR this study pmCUPZuoG33W pmCUP313-Zuo G33W, ampR this study pmCUPZuoP31A/
His6-SUMO-Ssz C81S C86S -Zuo C167S A343C, kanR
this study
5.1. Materials 5.1.7. Primer
Table 5.4.: Primer used in this study, relevant bases are indicated by lower cases
Primer Sequence(5’ → 3’)
pSUMOZuo-dN62 5’BsmBI CCAGTGcgtctcAGGTGGTATGACCGTTGatgAATCCAATGTC-GACCC
pSUMOZuo-dN62 3’XhoI CCAGTGctcgagtcaCACGAAGTAGGACAACAAGCTG
pmCUPZuo-dN62 5’BamHI cgggatccATGGGCTCTCATCACCATCATCACCATGGCTCTatgTTTT-CTTTACCTACC
pmCUPZuo 3’NotI CGCGCgcggccgctcaCACGAAGTAGGACAACAAGC
pmCUPZuo 5’BamHI cgggatccatgTTTTCTTTACCTACCC
Zuo H128C forw CAAGTTGTCAAGTACtgTCCAGACAAGC
Zuo H128C rev GCTTGTCTGGAcaGTACTTGACAACTTG
Zuo C167S forw GCTCAGTACGACTCATcTGATTTTGTTGC
Zuo C167C rev GCAACAAAATCAgATGAGTCGTACTGAGC
Zuo E30R forw CGTCCGGTCcgACCGGTTGGTAAGTTC
Zuo E30R rev GAACTTACCAACCGGTcgGACCGGACG
Zuo P31A forw CGTCCGGTCGAAgCGGTTGGTAAG
Zuo P31A rev CCAACCGcTTCGACCGGACG
Zuo G33W forw CGAACCGGTTtGgAAGTTCTTTTTGC
Zuo G33 rev GCAAAAAGAACTTcCaAACCGGTTCG
Zuo K341C forw GCAAAAGCTGACAAAtgtAAGGCTAAGGAAGC
Zuo K341C rev GCTTCCTTAGCCTTacaTTTGTCAGCTTTTGC
Zuo A343C forw GCTGACAAAAAGAAGtgTAAGGAAGCTGC
Zuo A343C rev GCAGCTTCCTTAcaCTTCTTTTTGTCAGC
Ssz C81S forw GCCATTTGACAAGTcTGATGTCAGC
Ssz C81S rev GCTGACATCAgACTTGTCAAATGGC
Ssz C86S forw GATGTCAGCAAGTcCGCTAACGG
Ssz C86S rev CCGTTAGCGGACTTGCTGACATc
5.1.8. Antibiotics
Ampicillin 100µg/ml (stock 100 mg/ml)
Chloramphenicol 25 µg/ml (stock 25 mg/ml)
Kanamycin 50 µg/ml (stock 50 mg/(ml)
All concentrations are final concentrations, stock solutions are filter-sterilized
5.1.9. Technical Equipment
Balance(s) Sartorius
Centrifuge (SS34) Sorvall
FastPrep MP
FPLC- ÄKTA Purifier GE Healthcare - Amersham
FPLC Workstation BioRad
French Press SIM AMINCO
L7-55 Ultracentrifuge Beckman
Mini UZ M150SE Sorvall
Multifuge 4 Heraeus
PCR BioRad
pH-Meter 766 Knick
QSTAR Pulsar i Hybrid MS/MS System Applied Biosystems/MDS SCIEX
Scanner Canon
Semi Dry Western Blot BioRad
Spectrofluorimeter Jasco
Spectrophotometer Ultraspec 3100 pro Amersham
Starion FLA 9000 FujiFilm
Vivaspin column 500, 10 kD MWCO Satorius stedim
5.1. Materials
2x Laemmli 100 mM Tris-HCl pH 6.8
4 mM EDTA
5x Laemmli 250 mM Tris-HCl pH 6.8
10 mM EDTA
HDX-KII buffer 25 mM HEPES-KOH pH 7.4
50 mM KCl 5 mM MgCl2
HDX-Quench-buffer 0.4 M K-Phosphate pH 2.2 HEPES - Low Salt (HEPES-LS) 40 mM HEPES pH 7.4
100 mM K-Ac 5 mM MgCl2 5% (v/v) Glycerol
2 mMβ-Mercaptoethanol HEPES - High Salt (HEPES-HS) 40 mM HEPES pH 7.4
1 M K-Ac 5 mM MgCl2 5% (v/v) Glycerol
2 mMβ-Mercaptoethanol HEPES - non reducing Low Salt 40 mM HEPES pH 7.4
100 mM K-Ac 5 mM MgCl2 5% (v/v) Glycerol HEPES - Elutionbuffer 40 mM HEPES pH 7.4
100 mM K-Ac 5 mM MgCl2 300 mM Imidazole 5% (v/v) Glycerol
2 mMβ-Mercaptoethanol Power Stainer 0.5 % (w/v) Coomassie R250
50 % (v/v) methanol 10 % (v/v) acetic acid Power Destainer 45 % (v/v) methanol
10 % (v/v) acetic acid
5.1. Materials 10 mM MOPS pH 7.5
15 % (v/v) Glycerol SDS -stacking gel buffer 4x 0.5 M Tris-HCl pH 6.8
0.4 % (v/v) SDS (20%) SDS -running gel buffer 4x 50 mM Tris-HCl pH 8.8
0.4 % (v/v) SDS (20%) SDS sample buffer 5x 1.5 M Tris-HCl pH 6.8
2 mM EDTA
1% (v/v) SDS (20%)
1% (v/v) β-Mercaptoethanol 10% (v/v) Glycerol
Bromophenolblue SDS -running buffer 25 mM Tris
0.2 M Glycine
silver-stain 0.2 % (w/v) g AgNO3
0.075 % (v/v) formaldehyde (37%) silver-developing 6% NaCO3 *H2O
0.015 % (v/v) formaldehyde
TBS 10x 100 mM Tris-HCl pH 8.0 1.5 M NaCl
TBS-TT 10x 100 mM Tris-HCl pH 8.0
1.5 M NaCl
5 % (v/v) Tween-20 Western Blotting Buffer 2.5 M Tris
2 M Glycine 0.5 % (w/v) SDS pH 8.0
5.2. Microbiological and Molecular Biological Methods
5.2. Microbiological and Molecular Biological Methods
5.2.1. Cultivation and Conservation of E.coli Strains
Strains were streaked out from permanent cultures to form single colonies on LB agar plates supplemented with the appropriate antibiotics. Plates were incubated at 37◦C for 12-16 h, if not stated otherwise, and stored at 4◦C. Single colonies were used to inoculate liquid LB medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator.
Each liquid culture was inoculated with a single cell colony and grown at 37◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD600). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO.
The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80◦C.
5.2.2. Cultivation and Conservation of Yeast Strains
S.cerevisiae strains were streaked out from permanent cultures to form single colonies on YPD plates or the appropriate selection plate when containing a plasmid. Plates were incubated at 30◦C for 2-3 days, if not stated otherwise, and stored at RT. Single colonies were used to inoculate liquid medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator. Each liquid culture was inoculated with a single cell colony and grown at 30◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD600). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO. The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80◦C.
5.2.3. Plasmid DNA Preparation
To purify plasmid DNA the QIAprep Spin Miniprep Kit (Qiagen) was used according to the protocol provided by the manufacturer.
5.2.4. PCR Mutagenesis
Using PCR technique various DNA-fragments can be amplified. A standard PCR reaction contained 1x reaction buffer, 1 µl of DNA template (50 - 100 ng), 100 µM of dNTP mix, 0.5µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for a standard PCR are shown in Table 5.6.
Table 5.6.: Cycling Parameters for PCR Segment Temperature Time
Table 5.7.:Cycling Parameters for single site directed mutagenesis
The Single Site-Directed Mutagenesis can be used to introduce single site mutations into a DNA template using polymerase chain reaction (PCR). This technique was used to generate the various cystein mutations in Zuotin. A standard ssm-PCR reaction contained 1x reaction buffer, 1 µl of DNA template, 100 µM of dNTP mix, 0.5 µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for the mutagenesis are shown in Table 5.7. The reaction products were treated with the restriction endonucleaseDpnI at 37◦C overnight, to digest the parental DNA template. The DpnI endonuclease with the target sequence: 5’-Gm6ATC-3’ is specific for methylated and hemimethylated DNA (Nelson et al., 1992). Subsequently the multiply mutated single stranded DNA was transformed into DH5α competent cells.
5.2.6. Restriction Digest
Restriction digests were performed using the corresponding restriction endonuclease. 5 - 20µl of DNA sample (plasmid or PCR product) were incubated with 3µl 10 x reaction buffer and 0.2 - 1 U of the corresponding enzyme (w/o BSA) in a total volume of 30µl.
5.2. Microbiological and Molecular Biological Methods After incubation at the enzyme-specific temperature for 5 - 16 h the digest was used
for further work.
5.2.7. Ligation
Ligases link DNA ends by catalyzing the phosphodiester-bonding. DNA fragments with matching digested ends were incubated in no more than 10 µl total volume with 40 U T4-DNA-ligase and the recommended ligation buffer. The optimal ratio of vector to insert DNA was 1:3 for “sticky end” ligations. Different conditions for incubation were used: over night at 16◦C, 2 h at room temperature or 30 min at 30◦C.
5.2.8. Preparation of Chemically Competent E. coli Cells
A 50 ml culture was inoculated with 0.5 ml of an overnight culture and grown in medium (LB broth with 20 mM MgSO4, 10mM KCl) to mid-logarithmic phase. The cells were kept on ice for 10 min, pelleted at 1,500g for 10 min at 4◦C, resuspended gently in 150 ml cold TFB1 and incubated for 15 min on ice. The cells were sedimented again at 1,500g for 10 min at 4◦C and resuspended in 20 ml cold TFB2. The cells were aliquoted in 250 µl portions, frozen in liquid nitrogen and stored at -80◦C.
5.2.9. Transformation of Chemically Competent E. coli Cells
For transformation, 1-5 µl DNA were incubated with 50µl competent cells for 30 min on ice, then the cells were heat-shocked for 90 s at 42◦C and subsequently kept on ice for 2 min. 1 ml of LB medium was added and the mixture was shaken for 45 min at 37◦C to allow bacterial recovery and expression of the antibiotic resistance gene.
The transformed cells were plated onto LB agar plates containing the appropriate antibiotics.
5.2.10. Transformation of Yeast Cells
3 µl of 10 mg/ml boiled carrier DNA and 1 µg plasmid were mixed with 100 µl transformation mix (400 mM lithium acetate, 40 % PEG-3350, 130 mM β-Me). For transformation one large yeast colony was resuspended in the mix and vortexed. The mixture was then incubated rotating for 30 min at 37◦C. The transformed cells were
pelleted, resuspended in H2O and subsequently plated onto agar plates containing the selective media.
5.2.11. Spot Test
Yeast cells were grown to exponential phase and adjusted to OD600 = 0.2. Serial dilutions were spotted on agar plates containing selective media and appropriate aux-otrophy marker.
5.2.12. DNA - Gel-Electrophoresis / Agarose Gel
Agarose gel electrophoresis is used to separate DNA strands by size and can be utilize to estimate the size of the DNA. Negatively charged DNA molecules migrate through the agarose matrix using an electric field, with shorter molecules moving faster than longer ones. Usually 1% agarose gels prepared in 1xTAE electrophoresis buffer were used in a casting tray. One-sixth volume of a 6 x concentrated loading dye is added to each sample, mixed and loaded into the wells. The samples are separated at 50 -100 V (depending on the size of the gel) until the required separation is achieved. To visualize and photograph the DNA fragments, a long wave UV light box is used.
5.2.13. DNA Sequencing
All mutations were verified by DNA sequencing performed at GATC - Biotech, Kon-stanz.
5.3. Protein Biochemical Techniques
5.3.1. Protein Expression in E.coli
To ascertain that the Zuotin mutant proteins are over-expressed and soluble, the ex-pression and solubility of the mutant Zuo proteinsin vivowere first analyzed. A single colony of the E.coli strain expressing the desired Zuotin variant was used to inoculate 5 ml LB kan/cm media to grow over night. A 1:100 dilution of the overnight culture was then grown at 37◦ to an OD600 of 0.6 - 0.7 and a 1 ml sample of the culture was taken (S1). Then the cultures were shifted to 30◦C and the protein expression was induced by addition of 0.1 mM IPTG. After 3 additional h of growth a second
5.3. Protein Biochemical Techniques 1 ml sample was taken (S2). The rest of the culture was collected (S3). To analyze
the expression level, samples S1 and S2 were analyzed by SDS-PAGE. To analyze the solubility of the overexpressed protein, the cells of sample S3 were harvested (16,000 x g, 3 min) and resuspended in HEPES-LS buffer. The cells were lysed using FastPrep (1ml cells + 1g acid washed beads). Finally, the sample was centrifuged (14,000 x g, 20 min, 4◦C) and the supernatant (soluble fraction) as well as the pellet (insoluble fraction) were analyzed by SDS-PAGE.
5.3.2. Protein Purification from E.coli
To analyze the specific function of a protein, the protein must be present in a pu-rified biologically active form. To purify proteins differences in size, shape, charge, hydrophobicity or solubility can be used. All proteins used in this study were overex-pressed from the vector pCA528 (pSUMO) (Andreasson et al., 2008b) and processed according to the following protocol. The proteins carry an N-terminal His6-SUMO fu-sion tag which increases solubility and can be removed by the specific SUMO protease Ulp1 without any remaining amino acids (Malakhov et al., 2004).
5.3.2.1. Cell Growth
To purify all proteins used in this study (Ssz, Zuo, RAC and its variants), a 50 ml over night culture (LB kan/cm) was used to inoculate 3 liter of LB kan/cm media.
Cultures were incubated at 37◦C with shaking until an OD600= 0.7 - 0.8 was reached.
Then the cultures were shifted to 30◦C and the protein expression was induced by adding 0.1 mM IPTG. After 3-4 h of incubation cells were harvested (4,400 rpm, 30 min, 4◦C). The pellets were stored at -20◦C.
5.3.2.2. Cell Lysis
To lyse the cells, the pellets were resuspended in ice cold HEPES-HS buffer in a final volume of 35 ml. Using a French press the cells were lysed using high pressure (10,000 psi). 1 ml of protease inhibitor cocktail mix (TMcomplete, 1 tablet dissolved in 1 ml water) and 1 ml PMSF (10 mg/ml in Isopropanol) were added after the first round of lysis and the French press was performed a second and third time. Then the lysate was centrifuged (15,000 rpm, 30 min, 4◦C) and the supernatant was stored on ice for further processing.
5.3.2.3. Nickel-Affinity Chromatography
The cell lysate was incubated with 5 g Ni-IDA-matrix (Protino, Macherey-Nagel) for 1 h at 4◦C. After incubation, the matrix was washed with buffer (2x HEPES-LS, 2x HEPES-HS and 2x HEPES-LS) before transferring to a disposable column. Bound protein was eluted with HEPES-Elutionbuffer (containing 300 mM imidazole). The used Ni-IDA matrix was cleaned and regenerated according to the protocol provided by the manufacturer.
5.3.2.4. SUMO-tag Cleavage and Removal
The eluted fusion protein was supplemented with Ulp1 protease (10µg/10mg eluted protein), which cleaved the HIS6-Smt3 tag and the mixture was dialyzed over night at 4◦C against HEPES-LS buffer.
5.3.2.5. Ion Exchange
Anion exchange chromatography is based on the ionic interaction of negatively charged proteins (the mobile phase) with immobilized positive charges of the column material (the stationary phase - ResourceQ). In cation exchange chromatography positively charged proteins interact with the negatively charged column material (Resource S).
To purify Zuotin (pI = 8.8) ResourceS columns (1 ml or 5 ml, GE Healthcare) and for Ssz (pI = 4,78) and RAC, ResourceQ columns (1 ml or 5 ml, GE Healthcare) connected to an ÄKTA FPLC were used . The columns were equilibrated with 5 column volumes HEPES-LS buffer. Then, the protein solution was applied to the column using a super loop. The column was washed with 10 column volumes HEPES-LS buffer. To elute the desired protein a linear gradient from low to HEPES-LS buffer was applied over 20 column volumes. 0.5 or 1 ml fractions were collected and stored at 4◦C.
To test for the presence of the desired protein in the individual fractions SDS-PAGE was performed on selected fractions. The fractions which contained the purest concentration of the desired protein were then pooled and aliquoted to a volume of 200 -250µl, frozen in liquid nitrogen and subsequently stored at -80◦C.
5.3. Protein Biochemical Techniques
Table 5.8.: Ion Exchange chromatography information Column Resource S, 1ml or 5 ml, capacity 25 mg or 125 mg
Equilibration 5 column volumes HEPES-LS buffer - 40 mM HEPES-KOH pH 7.4, 100 mM K-Acetate, 5 mM MgCl2, 5% glycerol, flow rate 1 -2 ml/min
Washing 10 column volumes HEPES-LS buffer - 40 mM HEPES-KOH pH 7.4, 100 mM K-Acetate, 5 mM MgCl2, 5% glycerol, flow rate 1 -2 ml/min
Elution 20 column volumes gradient - 40 mM HEPES-KOH pH 7.4, 100 - 1000 mM K-Acetate, 5 mM MgCl2, 5% glycerol, flow rate 1 - 2 ml/min
5.3.3. SDS PAGE
Sodium Dodecyl Sulfate Polyacrylamid Gel Electrophoresis (SDS-PAGE) is a tech-nique which admits to separate proteins under denaturing conditions according to their size.
To prepare the sample one-fifth of reducing 5x Laemmli buffer were added to the pro-teins and heated up to 95◦C, in order to destroy the secondary and tertiary protein structure. The samples were then applied on a 12% SDS Gel. A molecular weight standard was also applied onto the gel in order to estimate the size of the protein samples. Each gel was running at 25 mA and a maximum of 250 V.
5.3.4. Coomassie Staining
Proteins separated by SDS-PAGE are stained and visualized using Coomassie brilliant blue R-250 dye. The Coomassie dye binds to proteins through ionic interactions be-tween dye sulfonic acid groups and positive protein amine groups (especially arginine) as well as through Van der Waals attractions. Coomassie staining yields blue bands on a clear background, with a sensitivity of 0.05 - 0.1 µg/band.
Following electrophoresis, the gel was incubated in Power Stainer for 10 min while shak-ing. Subsequently the Stainer was removed and the gel was washed using Destainer
until discreet bands appeared. For documentation gels were scanned.
5.3.5. Silver Staining
Silver staining has a higher sensitivity compared to coomassie blue staining, visualizing protein amounts down to 1 - 20 ng per band. After SDS-PAGE the gel was incubated in fixation buffer for 45-60 min. The gel was then washed twice for 10 min with ethanol before pre-stained for 1 min in pre-stain solution. Subsequently the gel was stained for 20 min in the stainings solution. For developing the gel was incubated in developing solution until bands appeared. To stop the reaction the gel was incubated in stop-solution. For documentation gels were scanned.
5.3.6. Western Blotting - Semi Dry
Polypeptides can be specifically visualized using western blot analysis. After the sep-aration via SDS-PAGE the proteins are transferred onto an immobilizing membrane.
Therefore, the membrane and 4 pieces of Whatman paper were prewetted in Western Blotting buffer and then assembled in a sandwich. The transfer of the proteins was accomplished by applying 200 mAh per blot (mini gel) or 350 mAh (midi gel) for 1 h. The incubation of the membrane with the antibody solutions was accomplished as shown below:
Table 5.9.: Western blot - incubation schedule protocol Step Procedure
blocking 50 ml 5% milk powder in 1x TBS, shaken for 1h 1st antibody 50 ml antibody solution in 1x TBS, shaken for 1h washing rinsed with 1x TBS-TT
washed 3x 10 min with 1x TBS-TT
2nd antibody 50 ml antibody solution in 1x TBS, shaken for 1h washing rinsed with 1x TBS-TT
washed 2x 10 min with 1x TBS-TT washed 10 min with 1x TBS
5.3. Protein Biochemical Techniques To detect the secondary antibody which was coupled to an Alexa Fluor dye, Starion
FLA-9000 - FujiFilm image scanner was used.
5.3.7. Bradford Assay
The Bradford assay is used to determine the concentration of a protein solution (1 - 20 µg (micro assay) and 20 - 200µg (macro assay)). It works by the action of Coomassie brilliant blue G-250 dye, which specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues (Bradford, 1976). Coomassie binds to these residues in the anionic form, which can be monitored by its absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red).
5.3.8. BADAN Labeling
BADAN is a thiol-reactive probe that is environment sensitive, and therefore is used to monitor ribosome binding kinetics. To label protein with BADAN, different Zuo vari-ants were dialyzed against non-reducing HEPES-LS buffer to remove reducing agents such as β-Me. Subsequently, the samples were incubated with a 10 fold excess of BADAN (dissolved in DMSO) for 2 - 3 h at RT in the dark, to circumvent photo bleaching. Samples were then centrifuged and washed with HEPES-LS buffer in a vivaspin device prior to measurement. Upon covalent binding to proteins, BADAN fluorescence is changed in its emission maximum as well as by an increase in fluores-cence intensity, which can be easily used to check the labeling procedure.
5.3.9. Fluorescence Assay
Fluorescent dyes can be used to label proteins and thereby follow protein folding and unfolding as well as protein - protein interactions. Excited by photon energy (light) a fluorophore changes its energy state from the ground state S0 to its excited state S1. To relax back to the ground state the fluorophore emits a lower-energy photon, which is indicated by a longer wavelength. To monitor BADAN fluorescence emission of different Zuo variants, 2 µM of Zuo was incubated in HEPES-LS buffer at 25◦C in a cuvette in a fluorimeter equipped with a thermostat-controlled cell holder. The excitation wavelength was set to 395 nm. The emission wavelength was monitored
from 470 - 600 nm. Slit widths were set depending on the cuvette used.
5.3.10. MTSL Labeling
MTSL is a nitroxide paramagnetic spin label which is attached via a disulfide bond to a cysteine residue, enabling EPR measurements of the labeled site. To label Zuo H128C/C167S with MTSL, the protein was washed 5x with non reducing HEPES-LS buffer in a vivaspin device. Subsequently, a 3 fold excess of MTSL (in DMSO) was added and the probe was incubated for 1 h at RT and further o/n at 4◦C. The labeled protein was than washed 5x with non reducing buffer to remove free MTSL in a vivaspin device.
5.3.11. EPR Measurements
Measurements and data analysis of MTSL labeled Zuo was performed in collaboration with Dr. Malte Drescher at the University of Konstanz.
5.4. Mass Spectrometry
5.4.1. On-line LC/MS
In order to analyze the exact molecular mass of proteins by ESI-MS, the samples must be desalted from buffer before they can be eluted into the mass spectrometer. Protein molecular weights were determined by online LC/MS using a QSTAR Pulsar equipped with an electrospray ion source (Applied Biosystems/MDS SCIEX), two HPLC pumps (Agilent 1100 Series), a Rheodyne injection valve (Model 7725) with a 200µl stainless steel sample loop, and a 2-position/10-port valve with microelectric actuator (Valco C2-1000EP6) as described in (Rist et al., 2005a).
5.4.2. Partial Tryptic Digest
Purified wt Zuo (0.3 µg/µl), in the presence and absence of Ssz, was incubated with Trypsin (0.4 ng/µl) at 30◦C and 300 rpm. At several time points (10 s - 600 s) aliquots were taken and digestion was immediately stopped by adding 2x Laemmli buffer or
Purified wt Zuo (0.3 µg/µl), in the presence and absence of Ssz, was incubated with Trypsin (0.4 ng/µl) at 30◦C and 300 rpm. At several time points (10 s - 600 s) aliquots were taken and digestion was immediately stopped by adding 2x Laemmli buffer or