3. Materials and Methods
3.7. Protein refolding
Refolding reactions were done in collaboration with Michael Kerner and Dean Naylor. Protein refolding reactions containing chaperones (when present) were carried out with the following molar concentration ratios of chaperones to substrate: 1 substrate (monomer) : 2 GroEL (tetradecamer) : 4 GroES (heptamer) : 5 DnaK (monomer) : 2.5 DnaJ (monomer) : 2.5 GrpE (dimer). Chaperone- mediated refolding was stopped by complexion of Mg2+ with EDTA or CDTA,
which inhibits the ATPase activity of the chaperone. If, however, the subsequent enzymatic reaction for determination of the folding status was also inhibited by EDTA or CDTA, chaperone-mediated folding was stopped by quick hydrolysis of remaining ATP in the folding reaction with apyrase.
3.7.1. DAPA refolding
25 µM DAPA was denatured in 6 M GdnHCl in buffer A containing 10 mM DTT for 1 h at 25 °C and diluted 100-fold into buffer A containing 10 mM Na-pyruvate and 5 mM ATP in the absence or presence of chaperones as indicated. At specified time points, aliquots of the reactions were stopped with a final concentration of 12.5 mM CDTA. DAPA activity was determined colorimetrically as described (Vauterin et al., 2000). The assay buffer contained 200 mM imidazole pH 7.4, 35 mM Na-Pyruvate, 4 mM o-aminobenzaldehyde and 2 mM L-aspartate-β-semialdehyde (ASA, a gift from R. E. Viola and R. Moore, University of Toledo, Ohio, USA). The substrate ASA was stored in 4 M HCl at -20 °C and was neutralized with an equal volume of 4 M NaOH prior to usage.
3.7.2. DCEA refolding
DCEA was denatured with 6 M GdnHCl in buffer A containing 8 mM DTT for 1 h at 25 °C and diluted 100-fold (to 1 µM) into buffer A containing 15
µM pyridoxal 5-phosphate and 5 mM ATP in the absence or presence of indicated chaperones. At specified time points, aliquots (25 µl) of the different refolding reactions were stopped with 1 U apyrase. DCEA activity was measured at 37 °C in a coupled enzymatic assay, by following the production of NADPH
and corresponding increase in absorbance at 340 nm as described (De Biase et al., 1996).
3.7.3. ENO refolding
100 µM enolase was denatured in 6 M GdnHCl in buffer A containing 10 mM DTT for 1 h at 25 °C and diluted 100-fold into buffer A containing 5 mM ATP in the absence or presence of indicated chaperones. At specified time points, aliquots of the refolding reactions were stopped by transferring them to enzyme assay solution containing 50 mM Tris-HCl pH 8.1, 100 mM KCl, 1 mM 2- phosphoglyceric acid, 1 mM MgSO4 and 10 µM EDTA. ENO activity was measured essentially as described by (Spring and Wold, 1975); as a modification ENO activity measurements were stopped with 100 nM HCl to allow UV absorption at 230 nm.
3.7.4. GATD refolding
100 µM GATD was denatured in 6 M GdnHCl in buffer A containing 5 mM DTT for 1 h at 25 °C and diluted 100-fold into buffer A containing 50 µM MnCl2 and 5 mM ATP in the absence or presence of indicated chaperones. At
specified time points, aliquots of the reactions were stopped with 0.1 U/ µl apyrase. GATD activity was measured as described (Anderson and Markwell, 1982). The assay buffer contained 50 mM Tris, pH 8.2, 50 µM MnCl2, 5 mM NAD+ and 9 mM D-galactitol-6-phosphate. The substrate D-galactitol-6- phosphate was prepared by reduction of D-galactose-6-phosphate according to (Wolff and Kaplan, 1956).
3.7.5. METF refolding
METF concentrations were determined based on the absorption of bound FAD at 447 nm (ε=14300 M-1cm-1) (Sheppard et al., 1999). 50 µM METF was
denatured with 4.35 M GdnHCl in buffer A containing 10 mM DTT for 1 h at 25 °C and diluted 100-fold into buffer A containing 50 µM FAD, 1 g/L BSA and 5 mM ATP in the absence or presence of indicated chaperones. At specified time points, aliquots of the reactions were stopped by 40 mM CDTA. METF activity was measured at 25 °C utilizing an NADH-menadione oxidoreductase assay,
essentially as described (Sheppard et al., 1999). The assay buffer was 50 mM Tris pH 7.2, 2 mM EDTA, 1 g/l BSA, 180 µM menadione and 200 µM NADH.
3.7.6. METK refolding
METK was denatured for 1 h at 25 °C with 6 M GdnHCl in buffer A containing 8 mM DTT and diluted 100-fold (to 500 nM) into buffer A containing 5 mM ATP in the absence or presence of indicated chaperones. At specified time points, refolding reactions were stopped with a 26-fold molar excess of EL-D87K (GroEL-Trap) (Farr et al., 1997), which binds to non-native protein but due to an inhibited ATPase is unable to release it. METK activity was measured at 25 °C essentially as described (Markham et al., 1980) except that L-[35S]-methionine
(specific activity 50 Ci/mol) was used.
3.7.7. SYT refolding
50 µM SYT was denatured in 6 M GdnHCl in buffer A containing 10 mM DTT for 1 h at 25 °C and diluted 100-fold into buffer A containing 5 mM ATP in the absence or presence of indicated chaperones. At specified time points, 2 µl aliquots of the refolding reactions were transferred to 18 µl of an enzymatic assay reaction containing 20 mM MOPS pH 7.4, 100 mM KCl, 10 mM MgCl2, 10 mM NaF, 2 mM threonine, 5 mM ATP and 2 mM [32P]-PPi (0.5 MBq/µmol) at 37 °C
(Bullard et al., 2000). After 10 min, 2 µl aliquots were spotted onto PEI-cellulose plates and separated by thin layer chromatography using 4 M Urea, 0.75 M KH2PO4 as mobile phase. The formation of [32P]-ATP was quantified on a FLA-
2000 phosphoimager with Aida 2.31 imaging software.
3.8.
In vivo co-expressions
3.8.1. Co-expressions of chaperones and substrates in E. coli
BL21 (DE3) Gold cells, harboring either the pBAD33-ESL, pBAD33-EL, or pBAD33-MmES plasmids were transformed with individual pT7-substrate plasmids. Single colonies were picked and grown at 37 °C in LB medium with 0.1 g/l ampicillin (amp), 0.04 g/l chloramphenicol (cam), 0.2% glucose and 0.2% glycerol to OD600nm = 0.4. Chaperones were induced for 1 h by shifting cells from
glucose to arabinose (0.2%) containing medium. Control cells were grown continuously in LB with glucose. Following chaperone induction, the medium was changed back to glucose and supplemented with 1 mM IPTG for 1 h to induce expression of substrate proteins. Equivalent numbers of cells were taken for preparation of total, soluble and insoluble protein fractions. Cells were centrifuged and the material for total protein preparation was resuspended in SDS-PAGE sample buffer. The material for soluble/insoluble protein preparation was resuspended in lysis buffer (50 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.001% (w/v) Tween 20 and 0.4 mg/ml lysozyme), incubated on ice for 3 h and subjected to multiple freeze-thaw cycles in the presence of benzonase. Insoluble from soluble material was separated by centrifugation (20000 g, 30 min) and resuspended in SDS-PAGE sample buffer. Total, soluble and insoluble extracts were prepared in identical volumes to facilitate comparison. The levels of proteins were compared following 12% or 16% SDS-PAGE and Coomassie Blue staining.
3.8.2. Co-expressions of chaperones and substrates in S. cerevisiae
Bacterial proteins were expressed in S. cerevisiae YPH499 cells transformed with expression plasmids p415Gal under galactose promoter control grown in SC–Leu medium at 30°C. Protein expression was induced at OD600 = 0.5
with 2% galactose for 4 hrs. Spheroplasts were prepared by Zymolyase treatment and lysed in PBS containing 0.1% Triton X-100 and EDTA-free protease inhibitors (Roche). Samples were fractionated into soluble and pellet by centrifugation (20,000 x g for 15 min). Protein amounts were analyzed by immunoblotting. For GroEL/GroES co-expression with substrates, the above strain was co- transformed with substrate plasmid and GroEL (pSI215) and GroES (p426ADH) plasmids under copper and ADH promoter control, respectively, in SC–Leu– Trp–Ura medium. GroEL was induced with 0.5 mM CuSO4 for 3 hr before
induction of bacterial GroEL substrates. To examine bacterial substrate solubility in yeast ydj1-deficient background, the strain wy1 and its isogenic wild-type strain DS10 (Becker et al., 1996) were analyzed as above.