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1 Graduate Program in Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-3400, USA
2 The Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, New York 11794-3400, USA
3 Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA
(RECEIVED January 3, 2007; FINAL REVISION April 25, 2007; ACCEPTED May 1, 2007)
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Abstract |
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Keywords: enoyl reductase; InhA; fatty acid synthesis; isoniazid; Mycobacterium tuberculosis
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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Although INH has been the gold standard in anti-TB chemotherapy since its discovery in 1952, the CDC estimates that over 7% of new cases in the United States are resistant to this drug (Middlebrook 1952; World Health Organization 2003). INH resistance arises primarily from mutations in katG, which codes for a mycobacterial catalase-peroxidase enzyme that is responsible for drug activation (Zhang et al. 1992). While the ultimate target of activated INH has been the subject of much debate, it is clear that INH inhibits mycolic acid biosynthesis via effects on the fatty acid biosynthesis (FAS-II) pathway, leading to an accumulation of saturated hexacosanoic acid and cell lysis (Takayama et al. 1975; Vilcheze et al. 2000). Following activation, INH forms an adduct with NAD(H), and it is this adduct that inhibits InhA, the enoyl reductase in the FAS-II pathway (Zhang et al. 1992; Quemard et al. 1995). While mutations in inhA correlate with INH resistance, there is also evidence that INH forms a complex with KasA, the FAS-II 
-ketoacyl synthase condensing enzyme (Banerjee et al. 1994; Mdluli et al. 1996, 1998; Marrakchi et al. 2000; Slayden et al. 2000; Vilcheze et al. 2000; Larsen et al. 2002; Kremer et al. 2003). In addition, it has now been shown that a second INH-NADP adduct can inhibit other mycobacterial enzymes such as dihydrofolate reductase (Argyrou et al. 2006). Thus, it is likely that the in vivo effects of INH are complex. Since INH resistance arises primarily from a dysfunction in INH activation, compounds that inhibit the target(s) of INH but do not require activation are likely to be active against drug-resistant MTB. Consequently, understanding the exact mechanism of INH action is of critical importance.
While the majority of INH-resistant clinical isolates have mutations in katG, polymorphisms are also observed in inhA and kasA, supporting the proposals that these enzymes are targets for INH (Zhang et al. 1992; Dessen et al. 1995; Basso et al. 1998). The InhA mutations, such as Ile21Val, Ile47Thr, and Ser94Ala, cluster in and around the cofactor binding site, resulting in a large (20–160-fold) reduction in affinity of NADH for the enzyme (Dessen et al. 1995; Basso et al. 1998; Vilcheze et al. 2006). However, inhibition experiments with the INH-NAD adduct, which is a slow onset inhibitor of wild-type InhA with an overall Ki value of 0.75 nM, demonstrate that the InhA mutations have only a small effect on adduct affinity, thus questioning the relevance of the mutations to INH resistance. Given that the inhibition assays were performed on the isolated enzyme, we speculated that interactions between InhA and other proteins in the Mycobacterium might be critical for modulating the activity and inhibition of the enzyme. Indeed, there is increasing evidence that components of the FAS-II pathway associate with each other in vivo. Bloch and coworkers isolated a high MW fraction from Mycobacterium smegmatis cell lysate with FAS-II activity (Odriozola et al. 1977) that was subsequently shown to contain the FAS-II components InhA and MabA (Marrakchi et al. 2000, 2002). More recently, two-hybrid experiments have provided evidence for the existence of both heterotypic and homotypic protein–protein interactions within the members of the FAS-II pathway (Veyron-Churlet et al. 2004, 2005).
In order to determine whether the clinical inhA mutations affect InhA inhibition within the context of a multienzyme complex, we need to identify the interactions between the FAS-II components and determine how these interactions are affected by mutagenesis and/or enzyme inhibition. Based on the knowledge that the interaction of MabA, the FAS-II ketoreductase, with other FAS-II enzymes was altered when tetrameric MabA was converted to a dimeric species, we set out to determine whether the oligomerization state of InhA was affected by enzyme inhibition (Veyron-Churlet et al. 2004). In addition, we have also probed the effect of mutagenesis on both the oligomerization state of the enzyme and, using bacterial-two-hybrid methods, on the ability of InhA to interact with KasA. While initial cross-linking experiments raised the tantalizing possibility that inhibition resulted in dissociation of the InhA tetramer into dimers, more detailed studies, using analytical ultracentrifugation and size exclusion chromatography, now reveal that inhibition causes a conformational change in the enzyme. The relevance of these observations to INH drug resistance is discussed.
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Interactions between INH resistant InhA and KasA
The InhA-pTRG plasmids were mutated to encode Ile21Val, Ile47Thr, or Ser94Ala, in order to determine if the InhA mutations observed in INH-resistant clinical MTB isolates affected the ability of InhA to interact with KasA. Each of the mutants was screened with the KasA-pBT plasmid by cotransformation and evaluated as described above. All three mutant forms of InhA retained their interaction with KasA, with growth of colonies on selective and dual selective media comparable to that of wild-type InhA. The number and size of the colonies formed by the InhA mutants was similar to that of wild-type InhA, suggesting that the strength of the InhA-KasA interaction had not been affected by the mutations.
Cross-linking of InhA
Wild-type InhA
Using the chemical cross-linker bis(sulfosuccinimidyl)suberate (BS3), we cross-linked the wild-type InhA protein. Analysis by denaturing SDS-PAGE resulted in the detection of equal amounts of dimeric (61.34 kDa) and tetrameric (122.68 kDa) protein, with a less intense band of uncross-linked monomer (30.67 kDa) (Fig. 1). Our results are in agreement with previous reports that InhA is a tetramer (Rozwarski et al. 1999; Marrakchi et al. 2002). To evaluate the effect of INH on the oligomerization of InhA, the cross-linking experiment was repeated with INH-inhibited InhA. The inhibition was performed with the addition of NADH, MnCl2, and INH, to form the inhibitory INH-NAD adduct (Rawat et al. 2003). Controls lacking either INH or INH/MnCl2 were also performed. In contrast to the uncomplexed wild-type InhA, cross-linking of InhA inhibited with the INH-NAD adduct or bound to NADH cofactor with BS3 yielded only monomer and dimer bands on SDS-PAGE (Fig. 1).
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Lysine mutants
InhA contains nine lysine residues, three of which, Lys118, Lys132, and Lys165, can potentially be involved in cross-links across interface B in the InhA tetramer (Fig. 2B). The replacement of each of these lysines individually with arginine had no effect on cross-linking in the unliganded enzyme, which is seen as the retention of the tetramer (Fig. 4A). However, following reaction with BS3 the amount of tetramer was only substantially reduced for the Lys118Arg/Lys165Arg double mutant (Fig. 4B), providing evidence that multiple cross-links can form at interface B (Fig. 2B). This indicates that an 11 Å cross-link must be able to form between the Lys118–Lys165 or a combination of cross-links via Lys118–Lys132 and Lys165–132 residues across the B interface in the unliganded protein. Since these residues are separated by 26 Å or 23 Å and 19 Å, respectively, in crystal structures of InhA, ligand binding must cause a substantial movement of the two dimers with respect to each other.
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AUC–Sedimentation velocity (SV)
Using Sedfit, the MWs of free and ligand-bound InhA were all shown to be close to the expected MW for InhA tetramer (Table 1). However, in contrast to the SE analysis, the SV experiments revealed a clear change in the sedimentation coefficient(s) value for the enzyme upon ligand binding. Thus, while free InhA had an s-value of 5.82 ± 0.15 S, InhA bound to NADH or inhibited by the INH-NAD adduct had s-values of 6.32 ± 0.15 S and 6.33 ± 0.15 S, respectively (Table 1, Fig. 6).
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Discussion |
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Initially, therefore, we set out to evaluate whether the INH-resistant InhA mutations affected the ability of InhA to interact with other components of the FAS-II pathway. We chose to study the ability of InhA to interact with KasA, since InhA directly precedes KasA in the FAS-II pathway and since an interaction between InhA and KasA might explain how independent studies have suggested that both InhA and KasA are targets for INH. However, while the bacteria two-hybrid experiments showed the anticipated interaction between wild-type InhA and KasA (Veyron-Churlet et al. 2004), these studies failed to show any difference in the ability of the three InhA mutants to interact with KasA. In fact, the strength of the interaction appears unaltered by these mutations.
While the bacteria two-hybrid method provides qualitative information on the ability of two proteins to interact, the interacting partners are not necessarily present in their native oligomerization states, which could be important in the present context since InhA is a tetramer in solution while KasA is a dimer. Importantly, while our two-hydrid experiments were in progress, Veyron-Churlet et al. (2004, 2005) demonstrated that mutations at the dimer–dimer interface of MabA, the FAS-II -ketoacyl-ACP reductase, altered the ability of this protein to interact with KasA and KasB. Consequently, we designed a second set of experiments to directly assess whether inhibition of InhA by the INH-NAD adduct altered the oligomerization state of InhA, since this would likely affect InhA-dependent interactions in the FAS-II complex, and also whether the Ile21Val, Ile47Thr, and Ser94Ala mutations then altered the response of InhA to inhibition. Blanchard and coworkers had previously shown that the InhA mutants bind NADH cooperatively, in contrast to wild-type InhA, indicating that not only had the mutations altered the affinity of InhA for NADH, they had also affected communication within the InhA subunits (Basso et al. 1998).
In agreement with previous studies (Rozwarski et al. 1999; Marrakchi et al. 2002), cross-linking wild-type InhA with the homobifunctional chemical cross-linking reagent BS3 yielded bands on SDS consistent with tetrameric InhA, in addition to dimeric and monomeric forms of the protein. This reagent contains two N-hydroxysuccinimide esters, which are reactive with the primary amines of lysine residues, of which there are nine in each of the InhA monomers (Fig. 2C). Interestingly, when the same experiment was repeated on InhA inhibited by the INH-NAD adduct, only bands associated with dimeric and monomeric InhA were observed. A similar result was obtained when InhA was incubated exclusively with the cofactor NADH, suggesting that binding of cofactor or inhibitor to the tetrameric InhA has caused the protein to dissociate into dimers (Fig. 1). When we extended these studies to the three mutant InhA proteins, similar data were obtained in the presence of the INH-NAD adduct. However, even at saturating concentrations of NADH, cross-linking experiments indicated significant amounts of tetrameric InhA were present. The similarity in response of the three mutant proteins is consistent with our inhibition studies, which show very similar affinities of wild-type and mutant enzyme for the INH-NAD adduct (Ki 0.75–5 nM). The observation that tetrameric InhA can still be observed for the mutant enzymes in the presence of NADH may result from incomplete binding of the cofactor and/or incomplete occupancy of the InhA subunits. Alternatively, the cross-linking data may indicate that NADH binding populates a second conformational state in the mutant InhA proteins that is fundamentally different from the ligand bound population observed for the wild-type enzyme.
In order to further probe the effect of ligand binding on the structure of InhA, we performed an additional set of experiments using AUC and size exclusion chromatography. In contrast to the cross-linking data, these methods indicated that inhibited and cofactor-bound enzyme was still tetrameric. However, sedimentation velocity experiments did reveal an alteration in sedimentation coefficient for wild-type and mutant InhA proteins upon ligand binding (Table 1). Thus, one simple conclusion we can draw from all these data is that ligand binding causes a conformational change in the InhA tetramer that prevents one or more lysine pairs across a dimer–dimer interface from being cross-linked.
While there are several crystal structures of InhA in complex with a variety of ligands, there are no structures of uncomplexed InhA and, thus, no high resolution information on the structure of InhA before ligand binding. However, by analogy to AUC studies on the hexameric Helicobacter pylori VirB11 protein, which undergoes a 5%–7% increase in s-value upon binding ATP, we can conclude that the change in s-value observed for InhA upon complexation indicates that the protein structure has become more compact (Savvides et al. 2003). In order to provide more information on the nature of the ligand-induced conformational change, we performed a series of cross-linking experiments on InhA mutants in which we replaced candidate lysine residues with arginines.
Analysis of the ligand-bound InhA structure indicates that the protein contains two dimer–dimer interfaces, A and B (Fig. 2C). In order to cross-link the four monomers within the tetramer, lysine residues at both dimer–dimer interfaces must be within 11.4 Å of each other. Across interface A, only Lys8 and Lys240 are close enough to be cross-linked by BS3, as they are 9.8 Å apart (Fig. 2A; Rozwarski et al. 1999). Comparative MALDI analysis of the trypsin/Glu-C digests of the uncross-linked monomeric InhA and the cross-linked dimer reveals the generation of a mass peak of 2081, confirming the expected m/z for the reacted BS3 plus the two peptides containing Lys8 and Lys240 (Fig. 3). In contrast, no lysine residues were sufficiently close to each other to allow cross-linking between subunits at interface B. These data suggest, therefore, that the conformational change resulting from ligand binding causes one or more lysine pairs at the B interface to move apart, since observation of tetrameric protein on SDS-PAGE also requires formation of cross-links over interface B for the unliganded protein. To provide more information on the ligand-induced conformational change, we consequently replaced Lys118, Lys132, and Lys165 with arginine residues. While the single-lysine mutants all formed tetramer for unliganded enzyme on reaction with BS3, no tetramer could be observed for the Lys118Arg/Lys165Arg double mutant. Since Lys165 is involved in cofactor binding, the interaction of this residue with the NAD ribose can explain why ligand binding prevents this residue from participation in a cross-link. However, the fact that a cross-link can form between Lys118 in two different monomers, which are over 21 Å apart, indicates that these residues must be within 11 Å of each other in the unliganded protein. Thus ligand binding must cause a substantial movement of the two dimers with respect to each other. In order to provide further information on the ligand-induced conformational change, we are currently using X-ray crystallography to study the cross-linked InhA protein tetramer.
In summary we have provided direct evidence for a ligand-induced conformational change upon interaction of InhA with the INH-NAD adduct. However, while the modulation in InhA structure caused by enzyme inhibition may have a critical impact on the ability of InhA to interact with other components of the FAS-II pathway, similar changes in structure are also observed for the Ile21Val, Ile47Thr, and Ser94Ala InhA mutants. Currently, the only difference we are able to discern between wild-type and mutant InhAs is the observation that some fraction of the mutant InhA proteins form tetramers upon reaction with BS3 even in the presence of saturating NADH. However, the mutations have little effect on the affinity of InhA for the INH-NAD adduct and do not affect the ability of InhA to undergo the observed ligand-induced conformational change (Rawat et al. 2003). If these mutations arise from drug pressure, then they must modulate the antibacterial activity of INH. Since there is evidence that InhA interacts with other components of the FAS-II pathway in vivo, our current hypothesis remains that the mutations affect InhA inhibition and/or the consequences of this inhibition within the context of the FAS-II multienzyme complex. Alterations in the affinity of the adduct for InhA modulated via protein–protein interactions could also then affect the partitioning of the adduct between different enzyme targets within the cell, including the proteins identified recently by affinity chromatography (Argyrou et al. 2006). Further studies on the inhibition of InhA in the presence of other enzymes from the FAS-II pathway are currently in progress.
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Materials and Methods |
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Plasmid construction
PCR was used to amplify the kasA and inhA genes from the genomic H37Rv DNA using the following primers: inhA 5'-GCTCCTAGGATGACAGGACTGCTGG-3' (forward), 5'-ATAAGAATGCGGCCGCTTAGAGCAATTGGGTGT-3' (reverse). kasA 5'-ATAAGAATGCGGCCGCAGATGATTCAGCCTT-3' (forward), and 5'-ATGTCGAATTCCTCAGTAACGCCCGAAGGC-3' (reverse). PCR products were digested with the appropriate restriction enzymes and cloned in frame into either the bait plasmid, pBT, to produce fusion proteins with the DNA-binding domain, the full-length bacteriophage 
repressor protein, cI, or the target vector, pTRG, to produce hybrid proteins with the activation domain, the N-terminal of 
-subunit of RNA polymerase. The three InhA mutants, Ile21Val, Ile47Thr, and Ser94Ala, were constructed as previously described. All cloning/mutagenesis steps were performed in E. coli XL1-blue MRF cells (Stratagene).
Transformations
All protocols were adapted from the Stratagene BacterioMatch kit. BacterioMatch II Validation reporter competent E. coli cells were used for all interaction experiments. Transformations were performed in Falcon 2059 polypropylene tubes using a mixture containing 100 µL of cells, 1.7 µL of -mercaptoethanol, and 50–100 ng of plasmid (bait and target). Reactions were incubated on ice for 30 min (shaking gently every 10 min). Cotransformation reactions were then heat shocked at 42°C for 35 sec. After 2 min of incubation on ice, 900 µL of sterile SOC medium were added to each tube. SOC medium was prepared by autoclaving 20 g of tryptone, 5 g of yeast extract, and 0.5 g of NaCl to 950 mL of H2O, followed by the addition of 10 mL of 1 M MgCl2, 10 mL of 1 M MgSO4, and 2 mL of 20% (w/v) glucose, all filter sterilized. After 1.5 h of recovery incubation in a 37°C shaker bath, the cells were centrifuged at 700 g for 15 min at room temperature. Pellets were washed (twice) in 1 mL M9 His-dropout broth and centrifuged again for 15 min. Pellets were then resuspended in 600 mL of M9 His-dropout broth and incubated for an additional 2 h in a 37°C shaker bath. Cotransformants were plated on nonselective medium, for calculating the efficiency of the uptake of both bait and target plasmid, as well as selective and dual selective media for detection of positive interactions. Plates were incubated at 37°C for 1–3 d, to ensure the detection of both strong and weak interactions. All cotransformations were performed with both positive (pBT-LGF2/pTRG-Gal11P) and negative (pBT-LGF2/pTRG) controls. Positive control vectors pBT-LGF2 and pTRG-Gall1P produce the dimerization domain of the yeast Gal4 protein and a domain from a mutant Gal11 protein, respectively, which have been shown to interact in this strain of reporter E. coli.
Protein expression and purification
Wild-type and mutant InhA proteins were constructed, expressed, and purified as described previously (Parikh et al. 1999). Briefly, all InhA variant proteins were expressed with N-terminal hexa-histidine motifs in BL21 (DE3) pLysS cells (Novagen) and were subsequently purified by nickel affinity chromatography and immediately exchanged into 30 mM PIPES, 150 mM NaCl, and 1 mM EDTA (pH 6.8) by gel filtration using a Sephadex G-25 (Amersham Biosciences) column. Protein concentrations were determined using 
280 = 37.3 M–1 cm–1 for the wild-type, Ile21Val, Ile47Thr, and Ser94Ala proteins.
Cross-linking of the wild-type and mutant InhAs
The purified proteins were exchanged into the cross-linking reaction buffer (20 mM Na2HPO4, 0.15 M NaCl at pH 7.5) directly prior to the experiment. The cross-linking reaction contained varied concentrations (0.1–10 µM) of wild-type or mutant InhA and a 50-fold excess of bis(sulfosuccinimidyl)suberate (BS3) (Pierce) in 5 mM sodium acetate (pH 5.0). After allowing the cross-linking reaction to proceed for 30 min at room temperature, the reactions were quenched with 1 M Tris (pH 7.5) (final concentration 50 mM) and incubated for an additional 15 min. The cross-linked proteins were then concentrated and analyzed using 4%–20% gradient SDS-PAGE gels. Cross-linking reactions were also performed on inhibited InhA, prepared as described below, and in the presence of 150 µM NADH (Sigma).
Protein digestion and MALDI analysis
Three protein samples were extracted after SDS-PAGE analysis: wild-type InhA without BS3, wild-type dimer, and tetramer with BS3. Standard protein extraction and digestion with trypsin and Glu-C was performed. Briefly, protein bands were excised from the gel and washed overnight in 50% methanol (v/v) and 5% acetic acid (v/v). Unless otherwise noted, all steps were carried out at room temperature. Wash buffer was removed and a second wash was carried out for an additional 3 h. Gel pieces were then dehydrated with acetonitrile for 10 min and then dried thoroughly with a CentriVap. Samples were then reduced with 10 mM DTT for 30 min, followed by alkylation with 100 mM iodoacetamide for 30 min. Samples were then dehydrated with acetonitrile and rehydrated with 100 mM ammonium bicarbonate, twice. Trypsin (20 ng/µL) and endoproteinase Glu-C (200 ng/µL) were prepared in ice-cold 50 mM ammonium bicarbonate (pH 7.8). Dried gel fragments were rehydrated with trypsin and Glu-C for 10 min with vortexing, excess trypsin/Glu-C was removed, and digestion was carried out overnight at 37°C. Peptides were then extracted from the gel with 50% acetonitrile (v/v) and 5% formic acid (v/v). The volume of the extract was reduced and adjusted to 20 µL with 50% acetonitrile (v/v) and 1% TFA (v/v) in preparation for MALDI. Peptides were analyzed using a Bruker Daltronics AutoFlex II MALDI-TOF/TOF (Fig. 3). Peptide masses were calculated using the program MS-Digest (http://prospector.ucsf.edu/prospector/4.0.7/html/msdigest.htm). Intermolecular and intramolecular cross-linked peptide masses were assigned using the Automatic Spectrum Assignment Program (ASAP) (http://roswell.ca.sandia.gov/
mmyoung/asap.html).
Inactivation of wild-type and mutant InhA proteins by INH
Inactivation reactions were performed as previously described (Rawat et al. 2003). Briefly, each reaction contained 0.3 µM InhA, 300 µM INH, 1 µM MnCl2, and 150 µM NADH in 100 mM Na2HPO4 buffer (pH 7.5). After incubating for 2 h at room temperature, residual enzyme activity was assessed by monitoring the ability of the samples to oxidize NADH upon the addition of trans-2-dodecenoyl-CoA (Parikh et al. 1999; Rawat et al. 2003). The inactivation reactions were then prepared for cross-linking with BS3, as described above. Inactivated InhA was further purified by chromatography on G-25 to remove excess NADH, and analyzed using analytical ultracentrifugation (AUC) and gel filtration chromatography.
Structure-based mutagenesis
Lys118, Lys132, and Lys165 were all mutated to Arg using a standard mutagenesis protocol. Primers for the Lys118Arg mutation were 5'-TACGCGGATGTGTCCA G GGGCATCCACATCTCG-3' (forward) and 5'-CGAGATGTGGATGCCC C TGGACACATCCGCGTA-3' (reverse), and primers for the Lys132Arg mutation were 5'-TATGCTTCGATGGCCA G GGCGCTGCTGCCGATC-3' (forward) and 5'-GATCGGCAGCAGCGCC C TGGCCATCGAAGCATA-3' (reverse). All mutated nucleotides are designated in underlined bold. The Lys165Arg InhA mutation was available from previous studies (Parikh et al. 1999). In addition, the double-mutants Lys118Arg/Lys132Arg, Lys118Arg/Lys165Arg, and Lys132Arg/Lys165Arg were also constructed. All six InhA mutants were expressed, purified, and assayed as previously described (Parikh et al. 1999).
Analytical ultracentrifugation (AUC)
All protein samples were dialyzed into 20 mM Na2HPO4, 150 mM NaCl (pH 7.5) immediately prior to analysis. AUC experiments were performed at 20°C using a Beckman Coulter Optima XL-I analytical ultracentrifuge equipped with a scanning UV/VIS spectrometer to monitor absorbance during radial scans. Experiments were performed using a four-cell, An-60 Ti analytical rotor and either a six-channel, charcoal-filled Epon centerpiece (for sedimentation equilibrium, SE) or a two-channel, aluminum centerpiece (for sedimentation velocity, SV). For SE, samples were analyzed at three concentrations, 3 µM, 10 µM, and 30 µM and included free InhA, InhA plus NADH, and inhibited InhA. SE was performed at 18k rpm and the data were collected for 24 h or until successive scans at A280 nm remained constant. Partial specific volumes (0.7406) and solvent density (1.00727) were calculated using SEDNTERP software (www.bbri.org/RASMB/rasmb.html). Each data set was analyzed using HeteroAnalysis Version 1.0.102. For SV, samples with an A280 nm and A230 nm ranging from 0.5 to 0.9 were utilized. Data sets were collected for a minimum of 150 scans at a running speed of either 40k or 60k rpm. Each data set was analyzed using the Sedfit program (www.analyticalultracentrifugation.com) (Schuck 1998; 2000; Dam and Schuck 2004).
Gel filtration chromatography
A prepacked HiLoad 16/60 Superdex 200 column was equilibrated with 20 mM Na2HPO4, 150 mM NaCl (pH 7.5). Calibration of the column was performed using blue dextran to measure the void volume in combination with a Gel Filtration Standard kit (Bio-Rad). The data were fitted to the equation y = –3.24x + 5.95. After calibration, the column was reequilibrated with the same buffer and wild-type InhA samples varying in concentration from 3 to 120 µM were applied to the column, as well as inhibited InhA samples. Column effluent was monitored at A280 nm and plotted against the elution volume.
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Abbreviations: MTB, Mycobacterium tuberculosis; FAS-II, fatty acid synthase type two; INH, isoniazid; INH-NAD, isoniazid-NAD adduct; SE, sedimentation equilibrium; SV, sedimentation velocity.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062749007.
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References |
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