CP peptidase activity was determined by endpoint assay in 50 mM sodium phosphate buffer (SPB), pH 7.2, and 95°C (unless otherwise noted), with a microtiter plate reader (model HTS 7000 Plus Bio Assay reader; Perkin-Elmer, Wellesley, MA) by detection of 7-amino-4-methylcoumarin (MCA) released from the carboxyl terminus of N-terminally blocked peptides (Sigma-Aldrich, St. Louis, MO) (2
). Negative controls (no enzyme) were run in triplicate to account for thermal degradation of substrates. Kinetic constants were determined using a least squares fit of the appropriate model to the initial velocity (U/μg) as a function of substrate concentration (0.01 mM to 0.50 mM) at 95°C. One unit of protease activity was defined as the amount of enzyme required to release 1 pmol of MCA per min. Total protein concentrations were determined using the Coomassie blue dye-binding method (4
) (Bio-Rad, Hercules, CA) in microtiter plates with bovine serum albumin (Sigma-Aldrich, St. Louis, MO) as the standard.
Purification of 20S proteasome from P. furiosus grown at 80°C and 90°C.
P. furiosus (DSM 3638) was grown on sea salts medium (40 g/liter sea salts [Sigma-Aldrich, St. Louis, MO], 3.3 g/liter piperazine-N,N-bis(2-ethanesulfonic acid) buffer, 1 ml/liter trace elements, 5 g/liter tryptone, 1 g/liter yeast extract, 120 g total sulfur). The culture (12-liter working volume) was grown in a 14-liter fermentor (New Brunswick, Edison, NJ) at an agitation rate of 600 rpm, pressure of 0.5 bar, and sparge rate (N2) of 0.5 liter/min. The cells grew at 80°C (pH 6.2) for 16.0 h and then were shifted to 90.0°C and held at this temperature for 1 h. Culture samples of 2.4 liters were taken at 16 h and 17 h; the resulting cell pellet was stored at −80°C.
Purification of the proteasome from cells grown at 80°C and 90°C was done using the same purification scheme. The cell pellets were resuspended in 20 ml of 20 mM Tris, pH 8.0, passed (four times) through a French pressure cell at 16,000 lb/in2, and centrifuged (10,000 × g, 4°C) for 20 min. The soluble protein fraction was applied to a 40-ml Q-Sepharose XK 26/20 column (GE Life Sciences, Piscataway, NJ) and eluted between 0.5 and 0.7 M NaCl. Many of the smaller contaminating proteins were cleared from the resulting VKM-MCA active pool using a Microcon centrifugal concentrator of 100,000 molecular weight cutoff (MWCO). This pool was then applied to a hydroxyapatite (Calbiochem, San Diego, CA) XK 16/30 column. Elution occurred between 220 and 265 mM SPB during a linear gradient of 0.05 to 0.5 M SPB, pH 8.0. After concentration, this active pool was applied to a HiPrep Sephacryl S-300 high-resolution XK 16/40 column calibrated with a high-molecular-weight calibration kit (GE Life Sciences, Piscataway, NJ). All CP VKM-MCA activity was eluted in the first peak, which corresponded to a protein of approximately 660 kDa; these fractions were then concentrated using a 30,000 MWCO Centriplus centrifugal filter device (Millipore, Billerica, MD).
Cloning and expression of the P. furiosus CP genes in Escherichia coli.
The genes for the three proteins associated with the proteasome, psmA (α, PF1571; proteasome, subunit alpha), psmB-1 (β1, PF1404; proteasome, subunit beta), and psmB-2 (β2, PF0159; proteasome, subunit beta), were separately cloned into the pET-24d(+) vector (Novagen, Madison, WI). The α gene was amplified using the following primers: forward, 5′-TGAACGCCATGGCATTTGTTCCACCTCA-3′; reverse, 5′-ATAAAAATTGGATCCAAGTCAGTAGTTGCTATCCA-3′. The β2 gene was amplified with forward primer 5′-TTAGGTGGTGCTCATGAAGAAAAAGACTGGAA-3′ and reverse primer 5′-TAAGGAAGCCTGGATCCTTCATACTACAAACTCTT-3′. The β1 gene was cloned using an ORF that started with the fourth amino acid from the reported amino terminus (based on locations of the start codon and likely ribosomal binding site). N-terminal sequencing confirmed that the unprocessed β1 subunit was expressed correctly. The primers used for β1 gene amplification were forward, 5′-TGTTGCCCATGGAAGAGAAACTTAAGGGAA-3′, and reverse, 5′-AAATTGTCGGATCCTTGGACTACTTTAACATTTT-3′.
The α gene was expressed in E. coli BL21(DE3), while the β1 and β2 genes were separately expressed in E. coli BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA). Expression was induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (optical density at 595 nm, 0.60); cells were harvested 3 to 5 h after induction (37°C). The resuspended cell pellets were treated with lysozyme, sonicated (Misonix, Inc., Farmingdale, NY), and centrifuged (18,000 × g, 4°C) for 30 min. Two 20-min heat treatments of the soluble protein fractions for α and β1 were performed, the first at 85°C and the second at 90°C, to remove residual E. coli protein. Each treatment was followed by cooling on ice for 30 min and centrifugation (18,000 × g, 4°C) for 30 min to remove insoluble protein. The β2 protein preparation required only one 20-min heat treatment at 85°C.
Assembly of the recombinant CP.
To assemble the CP, the α and β proteins were combined in equimolar ratios to a final total protein concentration of 0.5 to 0.7 mg/ml. This mixture was then incubated at the indicated temperature for 1 h and cooled on ice for 1 h, and precipitated material was removed through centrifugation (16,000 × g, 4°C) for 30 min. The CP soluble protein was then purified by a gel filtration step to remove unincorporated α and β proteins, using the approach described above for the native CP. Fractions were concentrated using a 30,000 MWCO membrane filter, as described above for the native CP.
Cloning, expression, and purification of the P. furiosus PAN gene in E. coli.
The PAN gene (PF0115) was cloned into a pET-21b(+) vector (Novagen, Madison, WI) using the following primers: forward, 5′-GGTGATACATATGAGTGAGGACGAAGCTCAATTT3′; reverse, 5′-TAAAAATTAGGATCCTCAGCCGTAAATGACTTCA 3′. PAN was expressed using BL21-CodonPlus(DE3)-RIL (Stratagene) with induction by 0.4 mM IPTG (optical density at 595 nm, 0.60); cells were harvested 3 to 5 h after induction (37°C). Cells were resuspended in 20 mM Tris pH 8.0 plus 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM dithiothreitol (DTT), 1 mg/ml lysozyme and sonicated (Misonix, Inc., Farmingdale, NY). The samples were centrifuged for 30 min (18,000 × g, 4°C), and the supernatant was removed. The pellet was then resuspended in 20 mM Tris pH 8.0 plus 0.5% CHAPS, 1 mM DTT and heat treated at 85°C for 20 min. The sample was then cooled and centrifuged, and the pellet was then resuspended in the same manner as before and heat treated at 90°C for 20 min. The resulting suspension was centrifuged to remove precipitated debris, with the supernatant containing pure PAN.
2D gel electrophoresis of purified CP.
Purified samples of the proteasome (45 μg) were precipitated in a 10% trichloroacetic acid solution on ice for 1 h. The resulting pellet was washed three times with 150 μl of ice-cold acetone (−20°C) and then dried for 5 min at 60°C. The protein was resuspended in 125 μl of rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte ampholytes [Bio-Rad, Hercules, CA]) and applied to a 7.0-cm pH 4 to 7 isoelectric focusing (IEF) strip (Bio-Rad). The strip was subjected to active rehydration (50 V) for 16 h. The conditions used for focusing were as follows: 250 V, linear ramp of 20 min; 4,000 V, linear ramp of 2 h; 4,000 V, rapid ramp of 10,000 V-h. After IEF, the strips were incubated in equilibration buffer I (6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% sodium dodecyl sulfate [SDS], 20% glycerol, 2% DTT) for 10 min at room temperature, followed by another 10 min incubation in equilibration buffer II (6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, 2.5% iodoacetamide). For the second dimension, the IEF strip was then placed on top of a 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and covered with a two-dimensional (2D) agarose overlay gel (0.5% low-melting-point agarose, Tris base [2.9 g/liter], glycine [14.4 g/liter], SDS [1.0 g/liter]). The gels were stained with GelCode Blue staining reagent (Pierce, Rockford, IL) and analyzed on a GS-710 calibrated imaging densitometer (Bio-Rad).
Differential scanning calorimetry of recombinant P. furiosus proteasome and PAN.
The melting temperatures of all expressed proteins were determined using a CSC Nano differential scanning calorimeter (DSC; Calorimetry Sciences Corp., American Fork, UT). All samples were dialyzed against 50 mM SPB, pH 7.2, which was the buffer used to generate the baseline scan. Samples (0.21 mg/ml) were degassed and scanned from 25 to 125°C using a scan rate of 0.5°C/min for two heating and cooling cycles. Heat capacity-versus-temperature curves were generated using the software program accompanying the DSC instrument to determine melting temperatures. After each sample was analyzed on the DSC, activity assays and native gels were used to determine if complete or irreversible denaturation had occurred. Samples were centrifuged (16,000 × g, 4°C) to remove aggregates, total protein concentrations were determined, and activity assays were run simultaneously against the corresponding initial samples to obtain relative loss of activity.
Thermal inactivation of CP assemblies.
High-temperature incubation of the active recombinant proteasome forms (α+β2 and α+β1+β2 assembled at 90°C and 105°C) was used to compare their stabilities. Each assembly was adjusted to a baseline concentration of 0.15 mg/ml and incubated at 115°C in an oil bath for up to 12 h. Aliquots were taken at time points from 0 to 12 h and stored on ice until the end of the incubation period. The standard VKM-MCA microtiter plate assay was then used to compare the activities of the mixtures (300 ng enzyme, based on preincubation concentration, was mixed with 5 μM VKM-MCA and heated to 95°C for 15 min). The resulting fluorescence scores, with average background values subtracted, were determined and used to determine first-order decay constants.
Transcriptional analysis of dynamic heat shock response.
A whole-genome cDNA microarray including 2,065 ORFs was printed, following protocols described previously (5
). The array was used to determine the transient-transcriptional response after a temperature shift from 90°C to 105°C. P. furiosus
(DSM 3638) was cultured anaerobically at 90°C on sea salts medium (SSM), as described previously (37
). Tryptone (Sigma, St. Louis, MO) was added to SSM (final concentration, 3.28 g/liter) as a carbon source prior to inoculation, along with elemental sulfur (10 g/liter). A 60-ml batch culture was used to inoculate 500 ml of SSM supplemented with 3.28 g/liter tryptone and 10 g/liter sulfur in a 1-liter pyrex bottle. A 250-ml aliquot of this culture was added to 12 liters of medium in a 14-liter fermentor (New Brunswick Scientific, Edison, NJ). The fermentor contained an internal temperature controller, and the pH was maintained by a Chemcadet controller (Cole Parmer, Vernon Hills, IL). High-purity N2
was used to reduce the medium and to sparge during inoculation. The culture was grown to mid-log phase at 90°C, after which a sample was collected. The temperature set point was then shifted to 105°C, with the culture taking approximately 2 min to reach the set point temperature. Once the culture reached 105°C, samples were taken at 0, 5, 10, 60, and 90 min. Approximately 20 ml of culture was collected prior to sampling at each time point to eliminate preexisting fluid in the sampling lines. At each time point, 500 ml of culture was withdrawn and immediately put on ice until it was processed for RNA extraction. One ml of sample was removed for cell density enumeration by epifluorescence microscopy with acridine orange stain (20
RNA was extracted from each 500-ml sample culture as described previously (37
). The 500-ml samples from the fermentor were centrifuged for 20 min (10,000 × g
, 4°C). After treatment with RNA lysis buffer, the samples were stored at −70°C. Extractions proceeded with ethanol precipitation and purification using Ambion RNAqueous kits. Concentrations and degree of purity were determined by optical density at 260 nm and 280 nm, as well as with gel electrophoresis (1% agarose gel, 60 V). Procedures for reverse transcription reactions, aminoallyl labeling with Cy3 and Cy5, and hybridization reactions are reported elsewhere (5
A loop experimental design incorporated reciprocal labeling of time point samples with both Cy3 and Cy5. Mixed model analysis was used to evaluate differential expression data using approaches presented elsewhere (5
). Briefly, least squares estimates of gene-specific treatment effects, corrected for global and gene-specific sources of error, were used to construct pair-wise contrasts analogous to changes for each gene between all pairs of conditions. The statistical significance of these changes was determined, and a Bonferroni correction was used to establish an experiment-wide false-positive rate of α = 0.05 by dividing α by 2,821, the number of comparisons performed for all genes over all possible treatment pairs. The corrected false positive rate was 1.77 × 10−5
[corresponding to a −log10
) of >4.8]. Least squares estimates of gene-specific treatment effects were also used to perform hierarchical clustering in JMP 5.0 (SAS Institute, Cary, NC).