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MRC National Institute for Medical Research, London NW7 1AA, United Kingdom
(RECEIVED January 22, 2008; FINAL REVISION April 3, 2008; ACCEPTED April 7, 2008)
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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Keywords: Suf proteins; ATPase; hydrodynamics; kinetic mechanism
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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Our understanding of how individual Suf proteins interact, and what their primary roles are, lags behind that of the Nif and Isc systems. Here we have examined heterologously expressed Suf proteins from the thermophilic bacterium Thermotoga maritima so as to address an issue of central interest, namely how SufB, -C, and -D interact to form an active complex. The structures of SufC (Watanabe et al. 2005; Kitaoka et al. 2006) and SufD (Badger et al. 2005) have been solved. SufC is similar to the nucleotide-binding domain of ABC transporters, although there is no evidence that it functions as one. It has a unique Q-loop structure on the surface that has been postulated to be the binding site of SufD and/or SufB (Wilson et al. 2003; Watanabe et al. 2005; Kitaoka et al. 2006). SufD is a novel structure and forms a crystallographic dimer. Though the structure of SufB has yet to be solved and it has little sequence identity (
20%) with SufD, secondary structure predictions suggest it shares a similar fold.
Earlier observations showed that the desulfurase activity of the catalytic complex SufS/E in Escherichia coli is enhanced fourfold by interaction with the SufBCD complex (Outten et al. 2003). We and others have shown that SufC is an ATPase and that it interacts catalytically with SufB (Rangachari et al. 2002; Nachin et al. 2003, Eccleston et al. 2006). Our studies of the ATPase mechanism using 2'(3')-O-(N-methylanthraniloyl)- (mant) analogue nucleotides and transient kinetic techniques indicate that SufC initially binds ATP very weakly, followed by a conformational change of the SufC.ATP complex and then a slow hydrolysis step. ADP was bound to SufC more tightly than ATP and under our conditions remained bound (Eccleston et al. 2006). We expressed SufB, but it existed as a complex mixture of oligomers. This precluded quantitative kinetic studies of its effect on the SufC ATPase, though we were able to show that SufB accelerates the cleavage step of the hydrolysis reaction and reduces the dissociation rate constant of mantADP from SufC.
Here we have coexpressed SufB and -C, as well as SufC and -D, and purified the SufBC and SufCD complexes. We characterize them hydrodynamically and compare their interaction with fluorescent mant adenosine nucleotides with that of SufC alone.
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Results |
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120 kDa, they did not fit well into any of these models. Since the Edman degradation and quantitative SDS gel electrophoresis results showed that the complex contains equimolar amounts of SufC and SufD, we conclude that the complex exists in equilibrium between SufCD and SufC2D2. The decreasing apparent molecular weight on the trailing edge of the main peak of the light-scattering experiment is consistent with this interpretation (Fig. 3B). To investigate further, a solution of 140 µM SufCD was loaded onto an analytical gel filtration column and the elution profile monitored at 280 nm. Then successive fivefold dilutions were made of the SufCD, and gel filtration was repeated, the eluant being monitored at 230 nm at the lower concentrations to maintain a good signal to noise ratio. The complex eluted at increasing retention times with decreasing concentration, consistent with the equilibrium model. This equilibrium must be rapid on the timescale of our experiments.
Interaction of SufBC and SufCD with mant-nucleotides
The interaction of SufBC and SufCD with mant-nucleotides was investigated by kinetic methods. Although the hydrodynamic measurements described above showed that the complexes exist in solution as tetrameric species, for simplicity, the concentrations given are those calculated as if they were heterodimeric species. This is not important because in the single turnover experiments the concentration of the complexes is in excess of mantATP and so is saturating. The binding experiments were carried out with excess nucleotide under pseudo-first-order conditions, and so the rate constants are only dependent on nucleotide concentration.
Single turnover experiments of SufBC and SufCD with mantATP
Single turnover experiments were made in which 1 µM mantATP was mixed with 10 µM SufBC or SufCD in a stopped-flow instrument with excitation at 366 nm (Fig. 4). Data were collected in the "T" format and converted into total intensity and anisotropy. With SufBC, there was an increase in intensity of 80% over 2 s, which was accompanied by an increase in anisotropy from 0.06 to 0.23. This was followed by a decrease in intensity to 60% of the initial value and a decrease in anisotropy to 0.22 over the next 30 s. With SufCD, there was an initial increase in intensity, but only of 30%, and this was also accompanied by an increase in anisotropy from 0.06 to 0.20. Both intensity and anisotropy remained approximately constant over the next 100 s although there was possibly a very small exponential increase in intensity.
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The same reactions were repeated, and timed samples were quenched with acid, neutralized, and analyzed for the relative amounts of mantATP and mantADP (Fig. 5). With SufCD, there was an exponential decrease in the percentage of mantATP that could be fitted with a rate constant of 2.2 x 10–3 s–1. With SufBC, all of the mantATP had been hydrolyzed to mantADP by the time of the first time point (30 s). The reaction was therefore carried out in a quenched-flow instrument operated in the pulsed mode (Fig. 5B). It can be seen that there is an exponential loss of mantATP that could be fitted to a rate constant of 0.088 s–1. This corresponds to the slow decrease in intensity and anisotropy seen in the fluorescence data (Fig. 4). Since the rate constant of this process for SufC alone is 4.8 x 10–4 s–1, complex formation has accelerated this rate constant by 180- and fivefold for SufBC and SufCD, respectively.
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When 1 µM SufBC or SufCD was mixed with 10–50 µM mantATP, an apparently exponential increase in fluorescence intensity occurred (Fig. 6). However, the data showed some deviation from a single exponential fit. In the case of SufBC, the data were fitted to a single exponential plus a positive slope to take into account a small slow process following the initial process (see below). In the case of SufCD, the 10-µM and 20-µM data were fitted to a double exponential but the 30–50-µM data were well fitted to a single exponential. Observed rate constants obtained in this way were plotted against mantATP concentration. With both complexes, there was a linear relationship between k obs and mantATP concentration. This is consistent with this process being a second-order binding process. Under the pseudo-first-order conditions used here, the observed rate constant is given by
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where k 1 is the association rate constant and k –1 is the dissociation rate constant. Therefore, a plot of k obs against mantATP concentration has a slope giving the association rate constant and an intercept giving the dissociation rate constant. The second-order binding rate constants for SufBC and SufCD were 5.5 x 105 M–1 s–1 and 4.6 x 105 M–1 s–1, respectively. These are an order of magnitude faster than the rate constant for mantATP binding to SufC alone. Also, the intercepts were indistinguishable from zero, so a measurement of the dissociation rate constant could not be determined, unlike with SufC alone, with a dissociation rate constant of 3.8 s–1. However, the dissociation rate constants for the complexes are slower than this. Both the increased association rate constants and slower dissociation rate constants of the complexes result in the much tighter binding to them of mantATP than to SufC alone.
As mentioned above, following the second-order process, a smaller increase in intensity occurred that was difficult to determine on the timescale of these experiments. Because of their small amplitudes, it was difficult to determine the absolute values and whether or not they are dependent on concentration. The second step is ascribed to a conformational change of the SufBC.mantATP complex analogous to the one seen with SufC alone, which was accompanied by a larger signal and more easily defined. With SufCD, a small increase in intensity also occurred that was difficult to define in terms of amplitude and rate constant.
Interaction of SufBC and SufCD with mantADP
The kinetics of SufBC and SufCD binding to excess mantADP were studied under the same conditions as described above with mantATP. Again a rapid exponential increase in intensity occurred (Fig. 7A). This was followed by a second exponential process, but unlike with mantATP, it was well resolved from the first process both in rate constant and also in amplitude. Plotting k obs of the fast phase gave a linear relationship with the slope giving second-order rate constants for SufBC and SufCD of 9.1 x 105 M–1 s–1 and 6.9 x 105 M–1 s–1, respectively. Again the intercept was indistinguishable from zero. The rate constants of the second phase were 0.25 s–1 for both complexes and were independent of mantADP concentration over the range 10–50 µM.
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From the association and dissociation rate constants, a value for the equilibrium dissociation constants was calculated from the relationship Kd = k off/k on. For SufBC, this was 50 nM, and for SufCD it was 58 nM. This compares with a Kd of SufC alone of 2 µM. As with mantATP, mantADP binds much tighter to the complexes than to SufC alone.
Intrinsic fluorescence changes of SufBC and SufCD on interaction with ATP and ADP
SufC contains no tryptophan residues, but SufB and SufD contain seven and five tryptophan residues, respectively. This opened the possibility of using tryptophan fluorescence to explore the binding of ATP and ADP to the complexes without resorting to the use of fluorescent nucleotide analogs by exciting the tryptophan fluorescence at 280 nm and observing emission through a 320 nm cutoff filter.
Solutions of 1 µM SufBC were mixed with 10 µM to 1 mM ATP. A very small exponential decrease in fluorescence occurred (Fig. 8A). Over the range of 10–40 µM ATP, the rate constant of this process was linearly dependent on ATP concentration, with an association rate constant of 1.3 x 105 M–1 s–1 and an intercept indistinguishable from zero. When these experiments were repeated with ADP, there was again a small exponential decrease in fluorescence, and the second-order plot gave an association rate constant of 2.7 x 105 M–1 s–1. These data show that the signal arises from the binding of ATP and ADP to SufBC, and the association rate constants are comparable to those obtained with the mant-nucleotides.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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Hydrodynamic assessment of our complexes showed that SufBC exists in solution as a tetrameric species comprising two molecules of SufB and two molecules of SufC, i.e., SufB2C2, whereas with SufCD, the situation is more complex. We interpret our data to indicate a mixture in equilibrium between SufCD and SufC2D2, a conclusion supported by the decreasing molecular weight on the trailing edge of the multiangle light-scattering experiment where some SufCD would exist (Fig. 3B). This contrasts with the results of the same experiment with SufBC where the molecular weight remains constant across the peak (Fig. 3A). The increasing retention times on analytical gel filtration of serial dilutions also supports a tetramer–dimer model. The recently solved structure shows that SufCD is a crystallographic tetramer comprising two molecules each of SufC and SufD (K. Fukuyama, pers. comm.). Outten et al. (2003) isolated a complex of SufBCD from E. coli but did not determine its stoichiometry. We speculate that our engineered complexes, SufB2C2 and SufC2D2, are likely counterparts of the natural complex SufBC2D. The suggestion by Badger et al. (2005) that SufB and SufD might form a heterodimer based on their dimeric structure for SufD fits with this proposal.
A major finding of our kinetic experiments with SufBC and SufCD is that the initial binding of mantATP was much tighter to both complexes than to SufC alone. This resulted from a faster association rate constant and slower dissociation constant. The association rate constants for both complexes were identical within experimental error. With both complexes, following this binding step, a further small increase of fluorescence occurred that was difficult to quantify but that we ascribe to a conformational change that was more clearly defined with SufC alone. This was followed by the cleavage step—the rate constant was 180-fold faster for SufBC but only fivefold faster for SufCD, compared to SufC alone.
Binding of mantADP to both complexes again resulted in almost identical association rate constants, faster than with SufC alone. The binding step was followed by a well-defined further increase in fluorescence, with a rate constant independent of ADP concentration and that again we ascribe to a conformational change.
The results of the experiments with ATP and ADP and monitoring intrinsic tryptophan fluorescence were surprising since they obviously report on different steps of the ATPase mechanism with SufBC and SufCD. With SufBC, the rate constants are concentration dependent with both ATP and ADP and so report the binding step. The association rate constants are similar to those with mantATP and mantADP, showing that the presence of the mant group has not perturbed this process. With SufCD, the rate constant is independent of ATP or ADP concentration and so describes a first order step that we ascribe to a conformational change following the binding step, which is spectroscopically silent. This is consistent with the results with mantATP and mantADP, although with mantATP this process was ill defined.
In summary, the kinetic results show that for both complexes the binding of mantATP and mantADP is a second-order process, with similar association rate constants, and this is followed by a conformational change. The major difference between the complexes is that the subsequent cleavage step is accelerated much more in SufBC than SufCD compared to SufC alone. Possible reasons for this are described below.
The role of the ATPase of the Suf system remains unclear despite genetic studies showing that SufC can be essential (Nachin et al. 2003; Xu and Moller 2004). Outten et al. (2003) showed that AMPPNP had no effect on the activation SufES, but we have previously shown that mantAMPPNP binds to SufC very weakly (Eccleston et al. 2006). The effect of ATP on the Suf system in E. coli was not studied by Outten et al. (2003). Also, in work describing formation of [Fe-S] clusters on SufB, the effect of ATP on the process was not investigated (Layer et al. 2007).
The only available structure of a SufC.ADP complex showed that the glutamate residue of the Walker B motif is flipped away from the nucleotide-binding site (Watanabe et al. 2005), and so results in the low rate of the cleavage step unlike many nucleotide-bound structures, in some of which this glutamate residue interacts with a putative catalytic water molecule. In the recently solved structure of the SufCD complex, this glutamate points toward the ATP binding site (K. Fukuyama, pers. comm.). It could be that this movement is a part of the conformational change for which we have provided kinetic evidence, although why the acceleration of the cleavage step is much greater in SufBC compared to SufCD remains unknown in the absence of a SufBC structure.
Phylogenetic analysis of approximately 100 primary sequences of SufB and SufD (S. Sato, pers. comm.) showed that SufB is highly conserved (in two closely evolved forms), whereas SufD is not well conserved, though its secondary structure is likely to be. Conservation of the primary sequence of SufB suggests its molecular interactions have more rigorous requirements than SufD. Moreover, in E. coli only SufB carries a Fe-S cluster and only the SufBC complex interacts with SufE. However, the proposal (Layer et al. 2007) that the role of SufE in E. coli is to link SufS with SufBC cannot apply to T. maritima, where SufE is absent, unless SufU is its functional homolog. Indeed analysis of bacterial genomes suggests that subsets of the most complete suf operons are common. Speciation may have tailored the multifunctions of Suf proteins into discrete systems whose individual modus operandi will have to rely on individual investigations.
We have been interested to explain the continued presence of the Suf system in eukaryotic apicomplexan pathogens like Plasmodium, Toxoplasma, and Theileria. These parasites carry a relict non-photosynthetic plastid whose function is not fully understood, though biosynthetic pathways for fatty acid (FAS II), isoprenoid (DOXP), haem, tRNAs, and protein synthesis have been identified (Wilson et al. 1996; Waller et al. 1998; Jomaa et al. 1999; Sato and Wilson 2002). Some of the parasite plastid genomes encode SufB (Ycf24) (Ellis et al. 2001) as well as the other suf genes that persist as nuclear copies whose products are imported into the organelle (B. Clough, K.E.S. Ellis, K. Rangachari, S. Thakrar, S. Sato, and R.J.M. Wilson, in prep.). By contrast, the Suf pathway of Th. parva comprises only SufS and a putative SufE-fusion protein (Gardner 2005). In this case the Suf proteins might be dedicated to the formation of thio-tRNAs; indeed, it has been proposed that the raison d'etre for maintenance of relict plastids in non-photosynthetic plants and algae in general is for the generation of organellar tRNAs (Barbrook et al. 2006). By contrast, for parasites such as Plasmodium and Toxoplasma that carry the entire Suf system, we proposed (Wilson et al. 2003) that Suf functions in the plastid to assemble Fe-S clusters for imported apoproteins such as ferredoxin, lipoate synthase, and the isoprenoid biosynthesis enzymes LytB and GcpE. Recently, it was found that isoprenoids generated in the plastid are modified for the production of abscisic acid, an important signaling molecule implicated in the invasive life style of Toxoplasma (Nagamune et al. 2008). At present it is difficult to confirm directly any of these functions of Suf in apicomplexan plastids, as they have not been separated from mitochondria in subcellular fractions. Nevertheless, the absence of Suf proteins from humans and the ability of inhibitors of plastid housekeeping functions, e.g., doxycycline (Dahl et al. 2006), to kill these parasites suggests that the Suf system may provide a target for inhibitors in some major pathogens. To this end we are expressing the Suf proteins of M. tuberculosis for detailed examination and screening purposes.
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Materials and Methods |
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The PCR products were ligated into the pCR-Blunt cloning vector (Invitrogen). Silent mutations were then introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) to remove internal restriction sites within the sufB, -C, and -D sequences (see Supplemental material). All constructs were verified by DNA sequencing (Lark Technologies). Subsequently, sufB, -C, and -D genes were excised from the pCR-Blunt cloning vector and ligated into pET3aTr (Tan et al. 2005). The genes were then subcloned into the pST39 polycistronic expression vector (Tan et al. 2005). Several combinations of vectors were generated, including pST39-sufC-sufD and pST39-sufC-sufB (see Supplemental material). Other combinations were shown to be unsuccessful.
The recombinant plasmids were transformed into E. coli strain BL21(DE3)pLysS (Invitrogen). Bacterial colonies were grown in Luria-Bertani broth with ampicillin and chloramphenicol to maintain the pST39 and pLysS plasmids, respectively. SufC and SufD coexpression were induced with 1 mM IPTG at 37°C and an OD600nm between 0.5 and 0.7. Expression was continued for 3 h at the same temperature. SufC and SufB coexpression were induced with 0.2 mM IPTG at 18°C and an OD600nm between 0.5 and 0.7. Expression was continued for 12–18 h at the same temperature.
Protein purification
Similar purification strategies were applied to the different combinations of the SufC, -B, and -D polypeptides utilizing the poly-histidine tag on SufC.
Bacteria were harvested from 4-L cultures by centrifugation at 3300g and the cell pellets resuspended in 50 mL buffer A (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM dithiothreitol, 5% [v/v] glycerol with two tablets of protease inhibitor cocktail—Complete, EDTA free [Roche]). After sonication and ultracentrifugation at 3000g for 30 min at 4°C, the cleared 50-mL bacterial lysate was loaded onto a 12-mL Ni-NTA Superflow column (Qiagen) equilibrated in buffer A, at a rate of 1 mL min–1. The column was washed with buffer A until the absorption at 280 nm reached baseline. Bound proteins were then eluted stepwise with 25 mM, 125 mM, and 500 mM imidazole in buffer A and the protein peaks collected. The eluted complexes were resolved by NuPAGE 12% Bis-Tris SDS-PAGE.
When SufC and SufD were coexpressed, both fractions eluting at 125 mM (the major peak) and 500 mM imidazole contained SufC and SufD. However, analytical size-exclusion experiments (Superdex 10/300 GL, 24-mL bed volume, 100-µL injection, 0.4 mL min–1 at room temperature) showed that the fractions eluted with 500 mM imidazole contained soluble aggregates eluting in the void volume. Therefore, only the fraction eluting at 125 mM imidazole was used for subsequent purification.
The protein solution (30–50 mL) was dialyzed overnight against 20 mM Tris-HCl, pH 8, 300 mM NaCl, and 1 mM DTT and concentrated to <5 mL (Vivaspin 5 kDa or 10 kDa cutoff).
The final step of purification was size-exclusion chromatography on a Superdex 200 column (HiLoad 26/60 Superdex 200, bed volume 320 mL) equilibrated in 20 mM Tris-HCl, pH 8, 300 mM NaCl, and 1 mM dithiothreitol. The column was loaded with 2 mL of the protein solution and the complexes eluted under isocratic conditions at a flow rate of 1 mL min–1 (all steps were performed at room temperature). Three major peaks eluted: The first was at the void volume of the column, the second contained both SufC and SufD shown by SDS gel electrophoresis, and the third contained only SufC. Both the SufCD complex and the SufC were dialyzed against 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, and 1 mM dithiothreitol. Sample purity was estimated by NuPAGE 12% Bis-Tris SDS-PAGE, and the gels were stained with Coomassie Blue. For SufCD, purity was estimated by eye to be >95%, with a yield of 100 mg L–1 of culture.
When SufB and SufC were coexpressed, the complex was purified in exactly the same way as the SufCD complex and a similar purity obtained. The yield for SufBC was 60 mg L–1 of culture.
Concentrations of proteins were determined from their absorption at 280 nm using extinction coefficients of 75,875 M–1cm–1and 52,830 M–1cm–1 for SufBC and SufCD, respectively (ProtParam at www.expasy.ch), assuming a 1:1 stoichiometry.
Hydrodynamic measurements
Analytical ultracentrifuge equilibrium measurements were conducted with a Beckman Optima XL-A analytical ultracentrifuge (Beckman Coulter) equipped with absorbance optics using six sector cells. Three different concentrations of protein were used and allowed to come to equilibrium at 10,000, 12,000, and 15,000 rpm. All nine resulting data sets were globally fitted to a single species model using Beckman/Origin software. Sedimentation velocity measurements were made with the same instrument but using two sector cells. Solutions were centrifuged at 40,000 rpm and 200 scans at 280 nm were made at 5-min intervals. The data were analyzed as a distribution of sedimentation coefficients using Sedfit (Schuck 2000).
The solution molecular weights of SufBC and SufCD were determined using on line multiangle laser light scattering coupled with size exclusion chromatography (SEC-MALLS). Samples of the complexes (300 µg in 100 µL) were applied to a Superdex 200 10/300 GL column equilibrated in 20 mM Tris-HCl, 150 mM NaCl, and 1 mM TCEP at a flow rate of 0.5 mL/min. The column was mounted on a Jasco HPLC controlled by the Chrompass software package. The scattered light intensity of the column eluant was recorded at 16 angles using a DAWN-HELEOS multiangle laser light-scattering detector (Wyatt Technology Corp.). The protein concentration of the eluant was determined from the refractive index change (dn/dc = 0.186) using an OPTILAB-rEX differential refractometer equipped with a Peltier temperature-regulated flow cell, maintained at 25°C (Wyatt Technology Corp.). The wavelength of the laser in the DAWN-HELEOS and the light source in the OPTILAB-rEX were both 658 nm. The weight-averaged molecular weight of material contained in chromatographic peaks was determined using the ASTRA software version 5.1 (Wyatt Technology Corp.). Briefly, at 1-s intervals throughout the elution of chromatographic peaks the scattered light intensities, together with the corresponding protein concentration, were used to construct Debye plots (KC/R 
vs. sin2[/2]). The weight-averaged molecular weight was then calculated at each point in the chromatogram from the intercept of an individual plot. An overall average molecular weight was calculated by combining and averaging the data from the individual measurements.
Fluorescence measurements
Mant-nucleotides were synthesized, purified, and analyzed as described previously (Jameson and Eccleston 1997).
Rapid fluorescence measurements were made with a Hi-Tech-Scientific SF-61 DX2 stopped-flow instrument (TgK Scientific). Excitation was through a monochromator at 366 nm when the mant-fluorophore was directly excited or at 280 nm where the mant-fluorophore was excited by resonance energy transfer via tryptophan fluorophores. In both cases, emission was viewed through a Schott KV 399 cutoff filter. Intensity measurements were made with an "L" format whereas anisotropy measurements were made in the "T" format. In the latter case, the excitation light was polarized, and light emitted parallel and perpendicular to the plane of excitation was measured simultaneously. The data were converted to total intensity by the relationship I = I II + 2I 
and to anisotropy by the relationship A = (I II – I )/(I II + 2I ). Data were analyzed using Hi-Tech software. Slow time course measurements were made using an ISS PC1 fluorimeter with the same optical settings as the stopped-flow measurements. Data were analyzed using Grafit. For slow (>1 min) measurements of the time course of the cleavage of mantATP, 40-µL aliquots were taken at timed intervals and the reaction quenched by adding to 2.5 µL 10% perchloric acid and then neutralized by the addition of 1.75 µL 4 M potassium acetate. After removing denatured protein by centrifugation, samples were analyzed for the relative concentrations of mantATP and mantADP using HPLC monitoring mant fluorescence (Jameson and Eccleston 1997). For faster measurements, a Hi-Tech RGF-63 quenched-flow instrument (TgK Scientific) was used. For the stopped-flow and quenched-flow measurements, concentrations quoted are those after mixing and are therefore half of those in the syringes.
All hydrodynamic and kinetic measurements were made in a solution containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, and 1 mM dithiothreitol at 20°C.
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Electronic supplemental material |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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Footnotes |
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Reprint requests to: John F. Eccleston, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK; e-mail: jeccles{at}nimr.mrc.ac.uk; fax: 44-(0)-20-8906-4489.
Abbreviation: mant-, 2'(3')-O-(N-methylanthraniloyl)-.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.034652.108.
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