text:
The interaction of trimethylamine dehydrogenase and electron-transferring flavoprotein.

The interaction between the physiological electron transfer partners trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus has been examined with particular regard to the proposal that the former protein "imprints" a conformational change on the latter. The results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead caused by electron transfer from partially reduced, as-isolated TMADH to ETF. Prior treatment of the as-isolated enzyme with the oxidant ferricenium essentially abolishes the observed spectral change. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed. This is attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model for the interaction between the two proteins that involves "structural imprinting" of ETF.

The interaction between the physiological electron transfer partners trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus has been examined with particular regard to the proposal that the former protein "imprints" a conformational change on the latter. The results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead caused by electron transfer from partially reduced, as-isolated TMADH to ETF. Prior treatment of the asisolated enzyme with the oxidant ferricenium essentially abolishes the observed spectral change. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed. This is attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model for the interaction between the two proteins that involves "structural imprinting" of ETF.
Trimethylamine dehydrogenase (TMADH, 1 EC 1.5.99.7) isolated from the bacterium Methylophilus methylotrophus (isolate W 3 A 1 ) is an iron-sulfur-containing flavoprotein consisting of two identical, catalytically independent subunits. Each subunit has a molecular mass of 83 kDa and contains an unusual covalently linked 6-S-cysteinyl FMN cofactor (linking the flavin via its 6-position to Cys 30 of the polypeptide), a bacterial ferredoxin-type [4Fe-4S] center, and one equivalent of tightly bound ADP having an unknown function (1)(2)(3)(4)(5)(6)(7). The enzyme catalyzes the oxidative demethylation of trimethylamine (TMA) to dimethylamine and formaldehyde (presumably through an imine intermediate that spontaneously hydrolyzes once dissociated from the enzyme). This reductive half-reaction of the catalytic cycle results in two-electron reduction of the enzyme shown in Reaction 1.
The catalytic cycle is completed by an oxidative half-reaction that involves removal of reducing equivalents from the enzyme via its [4Fe-4S] center (after electron transfer from the FMN site) by electron-transferring flavoprotein (ETF), the natural electron acceptor for TMADH (1). ETF is a 63-kDa ␣␤ heterodimer containing one equivalent of FAD and one equivalent of AMP (like the ADP of TMADH, of unknown function) (8), and operates in a one-electron oxidation-reduction cycle between the oxidized FAD and the anionic semiquinone FAD . oxidation states (9). However, recently it has been shown that ETF can be readily reduced to the hydroquinone form by both trimethylamine and sodium dithionite reduction when it is bound to TMADH (10). The ETF semiquinone passes its reducing equivalent to an ETF:CoQ oxidoreductase and thus serves as an electron carrier between various catabolic enzymes and the principal respiratory chains of a variety of organisms (including man) (11,12). TMADH is an interesting example of a complex flavoprotein that possesses multiple redox-active centers and is a convenient system in which to study biological electron transfer. TMADH can take up a total of three electrons per subunit upon reduction: two at the FMNH 2 and one at the iron-sulfur center. The enzyme with two electrons per subunit (TMADH 2e Ϫ), the reduced form generated on reduction by trimethylamine, is not fully reduced and has two possible distributions: fully reduced FMNH 2 with oxidized [4Fe-4S] or flavin semiquinone and reduced [4Fe-4S]. The equilibrium between these two distributions is governed by the reduction potentials of the centers and is pH-dependent. At pH 7.0 the former distribution is favored by a factor of approximately two (13).
Previous studies have examined both intramolecular electron transfer within TMADH (13) and intermolecular electron transfer between TMADH and ETF (10, 14 -17). The former work has demonstrated that electron transfer from FMN to the iron-sulfur center in TMADH occurs rapidly, with k ET Ͼ1000 s Ϫ1 , and is not rate-limiting to overall catalysis. Electron transfer on to ETF has been studied by following the reaction of reduced TMADH with oxidized ETF, varying the concentration of the latter (14,15). These two studies have shown that the observed rate constant for electron transfer exhibits hyperbolic dependence on [ETF], from which an apparent limiting k ET of ϳ180 s Ϫ1 and a dissociation constant for binding of TMADH red and ETF ox of 10 M have been determined. Assuming a decay parameter ␤ for electron transfer as a function of distance of 1.4 Å Ϫ1 , this value for the limiting rate of electron transfer is consistent with a donor-acceptor distance of ϳ12 Å; approximately the extent to which the [4Fe-4S] is buried in TMADH. Tyr 442 lies at the surface of TMADH, constitutes one side of a shallow cleft on the surface of the protein nearest the buried [4Fe-4S] center (7), and it is likely that ETF docks close to Tyr 442 in forming the proteinprotein complex. Kinetic studies of the TMADH mutants Y442F, Y442L, and Y442G have shown that mutations at this site significantly influence electron transfer from TMADH to ETF; at 25°C, the Y442G mutant exhibits a ϳ30-fold decrease in the limiting rate constant for electron transfer and a 10-fold increase in K d (15).
Recently, the reaction of reduced TMADH with ETF has been reexamined (17), not only with wild-type enzyme but also several site-directed mutants at Tyr 442 , which had previously been investigated (15). Whereas results with the mutant TMADH forms were largely consistent with the earlier study, the wild-type enzyme was found to exhibit kinetics with a linear dependence on [ETF] rather than the hyperbolic dependence found in the two earlier studies. Based on this observation, a model was proposed in which mutations at Tyr 442 had very large effects on the rate of electron transfer from the iron-sulfur center of TMADH to the FAD of ETF, although the effect was masked by a rate-limiting conformational change with the wild-type enzyme. In subsequent studies (18), it was suggested that subsequent to this rate-limiting conformational change, ETF was converted to a form that was far more kinetically competent to be oxidized or reduced. This conformational change was proposed to persist for some time even after dissociation of ETF from TMADH, and the overall process was termed "structural imprinting" (18).
In an effort to resolve these conflicting interpretations in the literature and to clarify the kinetic behavior of the oxidative half-reaction of TMADH with ETF, we have further examined the spectroscopic changes and kinetics of their interaction. In particular, the protein concentration and oxidation state dependence of spectral changes associated with the formation of complex of TMADH and ETF have been reexamined. The results indicate that the spectral changes previously attributed to complex formation between TMADH and either ETF ox (10) or ETF sq (19) are instead attributed to electron transfer processes, which can be partially reversed under certain conditions. In addition to equilibrium studies of formation of the TMADH⅐ETF complex, kinetic experiments with wild-type and a Y442G mutant have also been reexamined. The results are fully consistent with the two earlier studies, and we find no evidence to support a model for electron transfer between TMADH and ETF that involves a rate-limiting conformational change within the TMADH⅐ETF complex or, by corollary, a profound effect on electron transfer upon mutating Tyr 442 . The results are also inconsistent with the formation of an imprinted state of ETF upon prior incubation with TMADH.

EXPERIMENTAL PROCEDURES
Chemicals-Mono-and di-potassium phosphate and TMA were obtained from Sigma. Sodium dithionite was from Virginia Chemicals. Ferricenium hexafluorophosphate was prepared as described by Lehman et al. (20,21). TOPO-TA (with TOP 10 electrocompetent cells) and pTrcHis2A were purchased from Invitrogen. All enzymes used in the molecular biology experiments (including NcoI and XhoI) were purchased from MBI Fermentas, except for Pfu DNA polymerase, which was purchased from Stratagene. All bacterial media were purchased from Fisher.
TMADH and ETF Purification-M. methylotrophus W 3 A 1 was grown on TMA as sole carbon source, and TMADH was purified as described by Steenkamp and Mallinson (1), with the exception that Sephacryl S-200 was used instead of Sephadex G-200 in the gel filtration step of the purification. The concentration of the purified TMADH on a per flavin basis was determined from the 442-nm absorbance of oxidized enzyme using a molar extinction coefficient of 27.3 mM Ϫ1 cm Ϫ1 (6). Routine enzyme assays were performed as described by McIntire (22). As-isolated TMADH is denoted here as TMADH ai to distinguish it from fully oxidized enzyme, obtained by treatment with ferricenium hexafluorophosphate. ETF was purified essentially as described by Steenkamp and Gallup (9). Again, Sephadex G-200 in the gel filtration step of the purification was replaced with Sephacryl S-200. Because ETF as-isolated was typically partially reduced, it was oxidized by reaction with ferricenium hexafluorophosphate prior to use (yielding ETF ox ). Residual oxidant was removed by passage through a Sephadex G-25 column equilibrated with 50 mM phosphate buffer, pH 7.0. The concentration of ETF was determined from the absorbance at 438 nm using an extinction coefficient of 11.3 mM Ϫ1 cm Ϫ1 (9).
Purification of Recombinant Trimethylamine Dehydrogenase-The gene encoding the previously reported TMADH Y442G mutant (15) was obtained by a modified version of the QuikChange mutagenesis protocol (Stratagene) using the wild-type gene cloned into the TOPO-TA maintenance vector (to be described elsewhere) and the mutagenic nucleotide: 5Ј-GGTGAGTGGAGCGGTCACCGCG-3Ј. The mutant gene was subcloned into the pTrcHis2A vector and the mutation confirmed by sequencing. This vector was used to transfect electrocompetent Escherichia coli BL21 DE3 cells and overexpress the recombinant protein.
For the recombinant wild-type and mutant enzymes, a rapid purification method was adapted and modified from the previously developed method for the purification of trimethylamine dehydrogenase from bacterium W 3 A 1 (1). Transfected E. coli BL21 DE3 cells from above were grown in 2 liters of TPP medium containing 0.1 g/liter flavin mononucleotide (FMN), 0.1 g/liter riboflavin, 0.2 g/liter ferrous ammonium sulfate, and 100 mg/ml ampicillin. Cells were grown to an A 600 ϳ0.6, induced with 400 M isopropyl-␤-thiogalactoside, grown for 24 h at 28°C, and harvested by centrifugation (7000 ϫ g for 10 min). Cells were resuspended in 50 ml of 50 mM potassium phosphate buffer, pH 7. Crude lysate from cell lysis/sonication was loaded onto a DEAE column and eluted using a linear 0ϳ0.5 M NaCl gradient. Fractions containing recombinant TMADH were collected and pooled based on purity (UVvisible spectrum, SDS-PAGE), and then concentrated. The enzyme was then loaded onto an S-200 column pre-equilibrated with potassium phosphate buffer and eluted with the same buffer. With this improved construct, increased levels of FMN incorporation were evident, typically reaching 70%. Enzyme concentrations were determined spectrophotometrically using an extinction coefficient of 27,300 M Ϫ1 cm Ϫ1 at 443 nm, after correction for the deflavo enzyme (6).
Binding Studies of TMADH ai and ETF ox or ETF sq -Solutions of TMADH were placed in a tonometer and made anaerobic by alternatively evacuating and flushing with O 2 -free argon. Using a Hamilton syringe, 1.2-ml samples of TMADH at the desired concentration were placed in one side of a split-cell cuvette that was sealed with a rubber septum and made anaerobic by flushing with O 2 -free argon. A solution of oxidized ETF (ETF ox ) of the same concentration was made anaerobic in the same way, and 1.2 ml of this solution was then placed in the other side of the cuvette. The stoichiometry of TMADH and ETF ox in each case was thus 1:1. A spectrum of the two proteins in separate compartments of the cuvette was then recorded using a Hewlett-Packard 8452A diode array spectrophotometer, after which the two compartments were mixed and a second spectrum taken. The [after-mixing] Ϫ [beforemixing] difference spectrum was then obtained mathematically using software provided by the manufacturer. Given the kinetically determined dissociation constant K d for the binding of the TMADH red and ETF ox of 10 M (14), a 10-fold greater concentration of the two proteins in the present experiment should ensure essentially complete formation of the protein complex (assuming the K d is not greatly affected by the oxidation state of TMADH). Because concentrations used in the experiments were limited by the light path of the cuvette and the nonlinearity of Beer's law at high optical density, the concentrations used here could be no higher than 100 M. The identical protocol was used to obtain the difference spectrum associated with formation of the TMADH ai and ETF red complex. ETF SQ was obtained by titration of ETF with sodium dithionite.
Simulation of Spectral Changes Associated with Electron Transfer from TMADH to ETF-The readily obtained spectra of TMADH ox , TMADH 2eq , ETF SQ , and ETF ox were used to calculate the difference spectrum of TMADH ox Ϫ TMADH 2eq and the difference spectrum of ETF SQ Ϫ ETF ox . These two difference spectra were then used to obtain a simulation of the spectral change associated with electron transfer from TMADH to ETF.
Treatment of TMADH with Phenylhydrazine Hydrochloride-Phenylhydrazine-inactivated TMADH (TMADH inact ) was prepared according to the method described previously (23): 50 M TMADH in 0.1 M sodium pyrophosphate buffer, pH 7.7, was incubated with 2 mM phenylhydrazine at 30°C for 4 h. The solution was then filterconcentrated and passed through a Sephadex G-25 column to remove excess phenylhydrazine. The concentration of TMADH thus obtained was determined using an extinction coefficient of 21.4 mM Ϫ1 cm Ϫ1 at 410 nm (6).
Kinetics of Electron Transfer between TMADH and ETF-Kinetic experiments were carried out using a Kinetic Instrument Inc. stoppedflow apparatus equipped with an On Line Instruments Systems (OLIS) model 3920Z data collection system, or an Applied Photophysics SX.17 MV stopped-flow spectrophotometer. Anaerobic native TMADH, TMADH inact , or mutant TMADH Y442G was made anaerobic in a glass tonometer by alternate cycles of evacuation and flushing with argon, and reduced by titration with sodium dithionite delivered via a titration syringe. Spectral changes were monitored via a side-arm cuvette attached to the tonometer. Fully oxidized ETF was prepared as described above and made anaerobic as above in a tonometer. The concentration of reagent in excess in the stopped-flow experiment (either ETF or TMADH) was at least five times greater than that of the limiting reagent to ensure pseudo first-order conditions. Kinetic transients obtained after mixing reduced TMADH with ETF were fitted to sums of exponentials using software provided by the manufacturers (24). Although well-established anaerobic procedures were used throughout (25,26), it is to be noted that the reduced forms of both TMADH and ETF are unusually unreactive toward oxygen (each requiring hours in air to become reoxidized). In addition, no evidence of oxygen contamination (e.g. a reduced absorbance changes seen in the course of an experiment) was observed in any of the studies reported here.

RESULTS
The Binding of As-isolated TMADH to Oxidized ETF and ETF Semiquinone-The spectral change observed upon mixing TMADH ai and ETF ox is shown in Fig. 1A (solid line) and is in good agreement with that reported previously (10). Previous studies of a variety of flavoproteins (27)(28)(29)(30)(31) have reported spectral changes similar to that seen here on mixing TMADH ai with ETF ox . Examples include the binding of aromatic amino acids or benzoate to D-amino acid oxidase (27,28), p-methoxybenzaldehyde to old yellow enzyme (29), tartronate to lactate oxidase (30), and 8-bromoxanthine to oxidized xanthine oxidase (31). These spectral changes have been interpreted to be arising from moving a normal non-covalently modified flavin from a more hydrophilic environment to a more hydrophobic one. Based on this earlier work, we have previously proposed (10) that the spectral change shown in Fig. 1A (solid line) is caused by a perturbation of the environment of FAD in ETF (rather than the spectrally distinct 6-S-cysteinyl FMN of TMADH) upon formation of the TMADH⅐ETF complex. If this is true, then it should be possible to use this spectral change to obtain a dissociation constant for ETF binding to (oxidized) TMADH. Fig. 1B shows the dependence of the observed spectral change on protein concentration, with results that are at least qualitatively consistent with a K d of ϳ10 M. The observed spectral change, however, also turns out to be very similar to the difference spectrum obtained by Huang et al. (14) for the oxidation of TMADH 3eq or of reduced phenylhydrazine-inactivated TMADH by ETF ox . This observation raises the question of whether the observed spectral change in the present experiment arises from complex formation between TMADH and ETF or from electron transfer processes.
In addition to the spectral change attributed to binding of TMADH to oxidized ETF, a very large spectral change associated with formation of a complex between as-isolated TMADH and ETF semiquinone (ETF sq ), with an extinction change of over 16.0 mM Ϫ1 cm Ϫ1 at 460 nm, has been reported (18,19). This extinction change exceeds the extinction coefficient for the anionic semiquinone at this wavelength, and the reported spectral change in fact closely resembles that seen upon reduction of TMADH. We have further examined the spectral change associated with mixing TMADH ai with ETF sq (reduced by titration with sodium dithionite), rather than the fully oxidized ETF ox . The observed spectral change is shown in Fig. 1A (dotted line). Surprisingly, this change is not at all like that reported previously (19) but rather appears to be the mirror image of that seen in the experiment using TMADH and ETF ox . It seems very likely that in the earlier study using ETF sq , the observed spectral change seen upon mixing TMADH ox with ETF sq was caused by overtitration of ETF with excess reductant, which reduced TMADH when the two protein solutions were mixed.
The observed mirror image relationship of the spectral changes seen for the (TMADH ai ϩ ETF ox ) experiment, on the one hand, and the (TMADH ai ϩ ETF sq ) experiment on the other, is extremely surprising if, as has been previously concluded, the former is in fact caused by a change in the environment of the FAD of oxidized ETF because the spectral signa- tures of FAD and FAD . are quite distinct. The question is thus raised as to whether the observed spectral changes might instead reflect electron transfer processes (from TMADH to ETF in the first experiment, and partial reversal, from ETF sq to TMADH, in the second) rather than complex formation per se. For this to be the case, however, it would require that asisolated TMADH be partially reduced, although perhaps to only a small degree. Similarly, electron transfer must be at least partially reversed (again possibly to only a small degree) in the second experiment.
Binding of TMADH ox and ETF ox -If the observed spectral change seen on binding TMADH ai to ETF ox is caused by electron transfer and as-isolated TMADH is indeed partially reduced, then prior treatment of TMADH with oxidant should eliminate the observed spectral change. Fig. 2 shows a comparison of the spectral changes upon binding ETF ox with as-isolated TMADH (from Fig. 1) or with oxidized enzyme (TMAD-H ox ) that had been pretreated with the oxidant ferricenium hexafluorophosphate. It is apparent that the extent of the observed spectral change is greatly decreased upon oxidization of TMADH, the difference spectrum being reduced essentially to the baseline. These results clearly indicate that as-isolated TMADH is partially reduced, although to only a small degree. From the absorbance change at 498 nm, and using a ⌬⑀ obs ϭ ⌬⑀ (TMADH ox Ϫ TMADH 2eq ) ϩ ⌬⑀ (ETF sq Ϫ ETF ox ) ϭ 5.4 ϩ 1.21 ϭ 6.61 mM Ϫ1 cm Ϫ1 (14), we estimate that 1.40 M reducing equivalents were initially present in the 40 M solution of TMADH ai . The implication is that the observed spectral change is in fact caused by electron transfer from partially reduced, as-isolated TMADH to ETF and not to a change in the environment of the FAD in ETF.
The difference spectrum for electron transfer between TMADH and ETF represents the sum of the spectra of the final states of enzymes (TMADH ox ϩ ETF sq ) minus those for the initial states (TMADH red ϩ ETF ox ), each species having a fully defined visible absorption spectrum. As a result, the expected spectral change for (TMADH ox Ϫ TMADH red ) ϩ (ETF sq Ϫ ETF ox ) can be calculated directly. This ⌬A is shown in Fig. 1C, where it can be seen that the calculated spectrum indeed provides a very good fit to the experimentally observed spectral change. This result supports the conclusion that the experimentally observed spectral change is a manifestation of interprotein electron transfer and not of complex formation, per se.
Kinetics of the Reaction of Reduced TMADH with ETF ox -On the basis of the above, it is not possible to determine a K d for the TMADH⅐ETF complex from absorbance changes seen with binding TMADH ox and ETF ox as a function of the concentrations of the proteins. The only reliable estimates for the K d for ETF binding to TMADH remain those determined kinetically from the [ETF] dependence of the rate constant for the reoxidation of TMADH red (14,15) and from ultracentrifugation experiments (32,33), which yield values for K d of 5-10 M. Although the concentration dependence of the reaction of reduced TMADH and ETF has been reported (14) and confirmed (15) to be hyperbolic in nature, recently a linear dependence for the wild-type enzyme has been reported (18). In order to further characterize the interaction of TMADH with ETF in light of the above, the kinetics of the reaction with ETF with two different reduced forms of TMADH (native TMADH and enzyme that has been inactivated by treatment with phenylhydrazine to render the flavin redox-inert, Ref. 6) have been reexamined. Previous studies have used excess ETF to react with limiting TMADH red . Although a wider range of concentration for ETF would be desirable, the strong absorbance of both TMADH and ETF and the need to work in an absorbance range where Beer's law applies significantly limits the experimentally accessible concentration range. As a result, we have performed experiments reversing the concentrations of the two proteins, using a pseudo first-order excess of TMADH rather than ETF. In both cases, the reaction can be followed conveniently at 370 nm, where the accumulation of the anionic semiquinone of ETF can be monitored.
Native or phenylhydrazine-inactivated TMADH was reduced by titration with sodium dithionite, following the course of the reduction spectrophotometrically. Fig. 3A shows a typical kinetic transient seen for the reaction of reduced TMADH inact (5 M) with ETF ox (50 M). Reduced TMADH inact has only one reducing equivalent in the iron-sulfur center, and because it can react with only a single equivalent of ETF, the overall reaction is greatly simplified. As seen previously (14), the observed transients are best fit as the sum of two exponentials, with k fast ϭ 128 s Ϫ1 and k slow ϭ 15.3 s Ϫ1 (accounting for 57 and 43% of the total observed spectral change, respectively); k fast for this and three other concentrations of ETF (under pseudo first-order conditions) are plotted in Fig. 3D (filled squares). Fig. 3B shows a transient for the reaction when the concentrations of TMADH and ETF are reversed, i.e. 50 M reduced TMADH inact and 5 M ETF ox . The transients are again biphasic, with the fitting results giving k fast ϭ 140.3 s Ϫ1 and k slow ϭ 45.3 s Ϫ1 (accounting for 61 and 39% of the observed ⌬A, respectively), in reasonable agreement with the experiment shown in Fig. 3A. Fig. 3C  the data obtained with pseudo first-order concentrations of TMADH are shown in Fig. 3D (filled circles). Although it is not technically possible to go to higher protein concentrations because of the strong absorbance of the flavin cofactor, overall the data from Fig. 3 are fully consistent with our previous results (13,15), which are reproduced along with our present data in Fig. 3D.
The data over the total concentration range that is experimentally accessible again show hyperbolic character for wild-type enzyme, not a linear concentration dependence with an intercept at the origin (19), with a K d of 10 M, and a limiting rate constant for electron transfer of 170 s Ϫ1 (Fig.  3D, solid line). The new data are not only quantitatively in agreement with the previous studies, but also specifically corroborate the asymptotic nature of the plot in the high concentration regime. Although the curvature in the combined data sets shown in Fig. 3D is readily evident, a linear fit to the data could be achieved if a non-zero y-axis intercept is allowed. Such a case would correspond to a simple one-step reversible process, with an association rate constant obtained from the slope of the plot and a dissociation rate constant from the y-axis intercept, with no formation of a discrete TMADH⅐ETF complex. In addition to the inferior linear fit to the data, we note that complex formation between TMADH and ETF is observed crystallographically as well as by ultracentrifugation studies (see below). We therefore do not favor the interpretation that the reaction proceeds in a single, reversible step.
To compare with native TMADH and re-check the role of Tyr 442 in the interaction of TMADH and ETF, we reexamined the concentration dependence of the mutant Y442G with ETF. As shown in Fig. 4, the mutant reacts more slowly than wildtype enzyme with ETF but again exhibits a hyperbolic dependence on [ETF]. Compared with wild-type enzyme, k obs is reduced ϳ40-fold to 4.0 s Ϫ1 and K d increased 8-fold to 80 M, consistent with both previous studies (15,18). It is evident that the mutation has a significant effect on the electron transfer kinetics, but the hyperbolic concentration dependence seen with wild-type enzyme is retained.
Further Assessment of the Imprinting Phenomenon-Based on both kinetic and fluorescence studies, it has recently been proposed that a rate-limiting conformational change in ETF within the TMADH⅐ETF complex occurs (18), and that ETF is subsequently converted to a structurally imprinted form. By this it is meant that binding induces a conformational change in ETF that makes it kinetically more easily reduced and which persists for a period of time even after dissociation from TMADH (18). TMADH is specifically proposed to catalyze the conversion of unimprinted to imprinted ETF. It has been suggested that such imprinted ETF differs structurally from asisolated ETF in only the immediate vicinity of FAD because overall molecular conformations did not show much difference by small angle x-ray scattering studies (25). That a conformational change of some sort must occur for formation of a TMADH⅐ETF complex is readily evident, because from their crystal structures the surfaces of the two proteins that undoubtedly must interact are both distinctly concave and do not complement one another. On the other hand, the properties attributed to the imprinted ETF itself are unusual. It is difficult to imagine, for example, how ETF maintains a metastable conformation even after dissociating from TMADH, particularly in light of the apparently highly dynamic nature of the ETF structure in solution (see below).
In order to critically evaluate whether TMADH catalyzes the conversion ETF to a structurally imprinted form, we have compared the kinetics of the reaction of excess native TMADH red with ETF ox , in the absence and presence of stoichiometric concentrations of TMADH ox in the ETF solution. If imprinting occurs, ETF in the latter experiment should be converted to its imprinted form, and significantly faster kinetics should be observed. The results of such an experiment (see Supplementary Materials) yield k fast ϭ 128.7 s Ϫ1 and k slow ϭ 1.3 s Ϫ1 without TMADH ox in the ETF solution and k fast ϭ 140.3 s Ϫ1 and k slow ϭ 0.79 s Ϫ1 in its presence. We note that the observed rate constants at the concentration of ETF used is consistent with the data shown in Fig. 3D. Although the second reaction is some 9% faster than the first, it is evident that the addition of TMADH ox to the ETF prior to reaction does not have the profound effect expected were ETF to have become imprinted. We conclude that TMADH does not catalyze the conversion of ETF to a new imprinted form that has significantly faster electron transfer kinetics. DISCUSSION The essentially mirror image relationship of the spectral changes seen on mixing TMADH ai with ETF ox on the one hand, and TMADH ai with ETF sq on the other, suggests strongly that these changes do not arise from a change in the environment of the (oxidized) FAD of ETF. Consistent with the interpretation that the observed spectral change is instead caused by interprotein electron transfer, the absorbance change is essentially abolished on pretreatment of as-isolated TMADH with the oxidant ferricenium prior to reaction with ETF ox . That the inverse spectral change is seen when TMADH ox is mixed with ETF sq indicates that electron transfer in the reverse of the physiological direction occurs (although to only a small degree) given the magnitude of the spectral change. In the TMADH ai ⅐ETF ox experiment (Fig. 1D). A comparison with the previous results of Huang et al. (14) indicates that the observed spectral change is indeed very similar to that expected for electron transfer, and only coincidentally resembles that associated with changes in the flavin environment.
In both the present work and in two previous studies of the oxidative half-reaction of TMADH, we observed saturating behavior in the reaction of TMADH red with ETF, with a hyperbolic dependence of the observed rate constant for electron transfer on the ETF concentration that yields a K d of ϳ10 M. A comparable value has been determined in ultracentrifugation studies, with a K d of 5 M obtained (32,33). As shown in Fig. 3D, the present kinetic results, using TMADH in excess as well as ETF, are fully consistent with our two previous studies (14,15).
The observation of a hyperbolic dependence of k ET on [ETF] with wild-type enzyme as confirmed here contrasts with the results of others (17) who have reported a linear dependence on [ETF] for wild-type TMADH (but saturating behavior for each of several mutants of TMADH). The apparent conversion of the electron transfer reaction from one of linear dependence of k obs on [ETF] into one having hyperbolic dependence has been incorporated into a model for the kinetic behavior of the system in which a conformational change within the TMADH⅐ETF complex is rate-limiting for electron transfer with wild-type enzyme, which is in turn assumed to be intrinsically very fast (17). The switch from linear to hyperbolic dependence on [ETF] going from wild-type to the mutants has been attributed to a very large effect on the intrinsic electron transfer rate k et . Mutations at Tyr 442 make the electron transfer process itself (rather than the proposed conformational change) increasingly rate-limiting as the side chain at position 442 is made progressively smaller. However, a model assuming an exceptionally high rate for electron transfer from wild-type enzyme, over a distance of at least 12 Å (reflecting the extent to which the [4Fe-4S] cluster is buried beneath the surface of TMADH) is not consistent with current views concerning biological electron transfer, where the polypeptide is assumed to represent a more or less uniform medium in which electron transfer occurs, with no single particularly effective pathway in operation (36) and (by corollary) no uniquely important amino acid residue. The effect of the Y442G mutation on electron transfer is certainly significant, with k obs ϳ4.0 s Ϫ1 and K d ϭ 80 M determined here for the mutant (Fig. 4) compared with the wild-type enzyme values of 172 s Ϫ1 and 10 M, respectively (15). The effect is not nearly so profound, however, as the orders of magnitude implied in the context of a model involving a rate-limiting conformational change within the TMADH⅐ETF complex, subsequent to which ETF becomes imprinted (17).
As indicated above, it is evident from an examination of the crystal structures of TMADH (37) and human ETF (which is highly homologous to the bacterial enzyme) (38) that the isolated proteins are not sterically compatible; both have surfaces that are concave in the vicinity of their respective redox-active centers (the [4Fe-4S] for TMADH and FAD for ETF). As a result, a conformational change in one or the other protein is required for the two redox-active centers to get close enough (Ͻ15 Å, Ref. 36) for effective electron transfer. Such a conformational change had in fact been previously suggested by Jang et al. (10) to account for the observation that M. methylophilus ETF can be fully reduced to the FAD hydroquinone form both enzymatically and chemically in the TAMDH⅐ETF protein complex. The idea of a conformational change in ETF in the course of electron transfer is thus not controversial. The present work calls into question, however, the suggestion that any such conformational change is rate-limiting for electron transfer from TMADH to ETF, and that subsequent to such a conformational change, ETF can be converted to a structurally imprinted state in which electron transfer is particularly facile. Much of the evidence in support of structural imprinting is based on spectral changes such as those examined here. Given our demonstration here that these reflect bulk electron transfer events rather than complex formation between TMADH ox and ETF sq , per se, the data in support of structural imprinting must be reconsidered. Further, the very similar kinetic behavior seen in the experiments involving reaction of excess native TMADH red with ETF ox with or without TMADH ox present in the ETF solution (see Supplementary Materials) demonstrate that the system does not behave as expected had ETF become imprinted. Specifically, were binding of TMADH to ETF to cause ETF to convert to a structurally imprinted form, much faster kinetics should have been observed in the experiment with TMADH ox present in the ETF solution than in its absence.
It is to be noted that the notion of structural imprinting as proposed for the TMADH/ETF system is also problematic from a thermodynamic standpoint. As formulated (18), the idea again is that as-isolated ETF exists in a conformation that is poorly disposed for electron transfer, but after transiently interacting with TMADH is converted to a form that exhibits more facile electron transfer properties. This form is proposed to persist for a significant period of time after ETF has dissociated from TMADH, prior to returning to the initial (and presumably more thermodynamically stable) unimprinted form. In order to comply with the First Law of Thermodynamics, this can only occur if the ETF, once converted to an imprinted state and dissociated from TMADH, only slowly reverts to the otherwise thermodynamically preferred unimprinted state in free solution. A thermodynamic box can be constructed linking the interconversion of an imprinted and unimprinted state for ETF and the binding of each to TMADH, as shown in Fig. 5. 2 Assuming that at equilibrium the imprinted state in as-isolated ETF is present below the levels of detection (say, 1%, reflecting a hypothetical equilibrium constant of 0.01 for formation of the imprinted state) and the same is true for the unimprinted state after exposure to TMADH (with a hypothetical equilibrium constant of 100), then TMADH must have a substantially greater affinity (10 4 in the present argument) for imprinted relative to unimprinted ETF. For the system to function as proposed, the rate constant for dissociation of the imprinted ETF from TMADH must be small, but also significantly larger than that for the thermodynamically favorable conversion of the imprinted ETF to the unimprinted ETF in free solution (otherwise, a catalytic amount of TMADH would be unable to quantitatively convert a solution of ETF from the unimprinted to the imprinted form, and/or the unimprinted state would never have formed in the first place). According to this argument, relaxation to the unimprinted state in free solution must also be slow.
Recently, the crystal structure of the TMADH⅐ETF complex has been reported (40), providing direct confirmation of its occurrence. Interestingly, electron density for the FAD domain of ETF is unresolved, indicating that it is disordered in the co-crystal, and very likely highly dynamic. We observe the same type of disorder in the FAD domain of ETF in the TMADH⅐ETF complex in our own co-crystals. 3 It is likely that this dynamic behavior allows ETF to accommodate complex formation with TMADH. A highly dynamic conformational flexibility in ETF is difficult to reconcile with the necessarily sluggish conformational changes required in the context of a structural imprinting model above, given the thermodynamic and kinetic constraints on the system. Similarly, it is unlikely that such highly dynamic conformational changes could be ratelimiting for electron transfer between TMADH and ETF. On the basis of this argument, and in conjunction with the kinetic work described here, we conclude that there is no imprinted form of ETF. 2 The thermodynamic box presented is fundamentally equivalent to the part of Reaction 4, presented in Ref. 18, relating to the formation of the imprinted state of ETF. Here, the spontaneous conversion of unimprinted to imprinted ETF in free solution is considered explicitly, however, as must formally be the case (even if the process is thermodynamically unfavorable). Also, in the present scheme the separate steps for formation of the TMADH⅐ETF complex and any subsequent conformational change to an electron transfer competent state are combined into a single macroscopic step, in light of the present results. It is to be emphasized that the equilibrium constant for this macroscopic step is simply the product of the equilibrium constants for the two individual steps, if they were to be considered discretely, and the argument presented remains valid. 3 W. W. Shi, C. Bell, and R. Hille, unpublished data.
FIG. 5. A thermodynamic box illustrating the linkage between imprinted and unimprinted states of ETF with binding to TMADH. TMADH has been proposed to catalyze the interconversion between the two ETF states (18). The hypothetical equilibrium constants for the two horizontal sides to the box are consistent with the properties attributed to unimprinted and imprinted ETF, as discussed in the text.


Speciation Begins, but Doesn't End, with the Twist of a Shell

1512 normal enzyme. Then, they replaced the oxygen in question with a sulfur atom. The reaction didn’t work as well because, by the rules of chemistry, sulfur doesn’t like to bind to magnesium. But sulfur does like manganese and cadmium ions. So they replaced the magnesium with one of these other metal ions and measured the reaction. The researchers saw that these other metal ions restored (or “rescued”) enzymatic activity. In short, the enzyme needs a bond where the oxygen and the magnesium are, but the bond doesn’t have to be between oxygen and magnesium. As complicated as that is, plucking out one atom and trading it for another is itself a tricky business. Because most enzymes are made of stubborn amino acids and not nucleotides, atomic mutagenesis can be diffi cult. And usually when researchers have tried atomic mutagenesis, they’ve mutated the substrate (the molecule that the reaction acts upon) instead of the enzyme (the molecule that acts). Here, Piccirilli, Herschlag, and colleagues directed the applications of atomic mutagenesis to the molecule that does the work. To test that a specifi c oxygen in the intron binds to the magnesium ion, the researchers fi rst had to compile a short list of potential atoms to which the magnesium might bind. By combining literature data from structural models and functional studies with a random sprinkling of sulfur atoms in the intron to fi nd critical oxygen contacts, Piccirilli, Herschlag, and colleagues established a group of specifi c oxygen atoms to watch. They tried the metal rescue experiment with each of these oxygens, and the only enzyme rescued by the metal switch was the one in which they changed the C262 oxygen to a sulfur. Therefore, they concluded that this specifi c oxygen atom makes a critical contact with the magnesium ion. The strategy of atomic mutagenesis combined with metal ion rescue can be used to help understand the mechanism of other RNA and protein metalloenzymes.

normal enzyme. Then, they replaced the oxygen in question with a sulfur atom. The reaction didn't work as well because, by the rules of chemistry, sulfur doesn't like to bind to magnesium. But sulfur does like manganese and cadmium ions. So they replaced the magnesium with one of these other metal ions and measured the reaction. The researchers saw that these other metal ions restored (or "rescued") enzymatic activity. In short, the enzyme needs a bond where the oxygen and the magnesium are, but the bond doesn't have to be between oxygen and magnesium.
As complicated as that is, plucking out one atom and trading it for another is itself a tricky business. Because most enzymes are made of stubborn amino acids and not nucleotides, atomic mutagenesis can be diffi cult. And usually when researchers have tried atomic mutagenesis, they've mutated the substrate (the molecule that the reaction acts upon) instead of the enzyme (the molecule that acts). Here, Piccirilli, Herschlag, and colleagues directed the applications of atomic mutagenesis to the molecule that does the work.
To test that a specifi c oxygen in the intron binds to the magnesium ion, the researchers fi rst had to compile a short list of potential atoms to which the magnesium might bind. By combining literature data from structural models and functional studies with a random sprinkling of sulfur atoms in the intron to fi nd critical oxygen contacts, Piccirilli, Herschlag, and colleagues established a group of specifi c oxygen atoms to watch.
They tried the metal rescue experiment with each of these oxygens, and the only enzyme rescued by the metal switch was the one in which they changed the C262 oxygen to a sulfur. Therefore, they concluded that this specifi c oxygen atom makes a critical contact with the magnesium ion. The strategy of atomic mutagenesis combined with metal ion rescue can be used to help understand the mechanism of other RNA and protein metalloenzymes.

Synopses of Research Articles
Open access, freely available online September 2005 | Volume 3 | Issue 9 | e310 | e317 Ribonucleic acid (RNA) is a dogma breaker. The "central dogma" of cellular biochemistry mandates that deoxyribonucleic acid (DNA) stores information, and RNA copies this information and uses it to direct the assembly of amino acid building blocks into proteins, such as enzymes. Enzymes catalyze important chemical reactions in the cell, such as the breakdown of glucose or the synthesis of urea.
When biochemists discovered catalytic RNA, they had to ditch the dogma. Because of its structure, it turns out, RNA can act as an enzyme and catalyze reactions. While two strands of DNA tend to zip up into the famous double helix, RNA usually goes solo. The single RNA strand folds back on itself to create myriad tangled arrangements. Some of these arrangements create an active center, the place on the RNA where the enzymatic magic happens. The many RNA enzymes and protein enzymes that use metal atoms to do their job are called metalloenzymes. One example of an important structural motif in RNA metalloenzymes is the group I intron, which can snip itself out of an RNA segment. Understanding exactly how the RNA and the metals interact will help to provide precise answers about how the enzyme really works.
Through X-ray crystallography, researchers have revealed many structural features of group I introns. But X-ray crystallography creates images of the enzyme frozen in time; it does not catch an enzyme in action. In a new study, Joseph Piccirilli, Daniel Herschlag, and colleagues discovered that a particular oxygen atom on a particular nucleotide in a group I RNA must bind to a particular magnesium ion in order for the reaction under study to proceed normally. The oxygen atom is known as the pro-S P phosphoryl oxygen at nucleotide C262 in the intron from the unicellular Tetrahymena thermophila protozoan.
Since there's no way to watch the oxygen and metal hook up during the reaction, how do the researchers know they do? The researchers used the powerful techniques of metal ion rescue and atomic mutagenesis. Here's how it worked. They fi gured out how well the group I intron reaction works with a The Ol' Switcheroo Shows How an RNA Enzyme Splices Itself DOI: 10.1371/journal.pbio.0030310 DOI: 10.1371 RNA enzymes called ribozymes require metal atoms to function. The site of metalribozyme interaction was studied by changing components of both the catalytic center of the ribozyme (the backbone) and its target substrate.
As warm-blooded creatures, humans and other mammals maintain a relatively stable body temperature that buckles under the stress of intense heat (or cold). When the heat gets too high, we develop fevers and weaken, and our proteins destabilize and degrade-in some cases, such reactions can prove fatal. But some organisms appear to defy nature (as we think of it) by fl ourishing in extremely high temperatures. The archaeal microbe Pyrobaculum aerophilum, for exampleoriginally found in a boiling marine water hole in Italy-thrives at ~100 °C (212 °F). Similarly, the bacterium Thermus thermophilus grows at temperatures between 48 °C and 85 °C (118-185 °F).
Such organisms are of interest for many reasons-not least of which is to understand the mechanisms that engineer their heat resistance, or thermostability. How do these thermophilic bacteria and archaea manage to maintain active, stable proteins at such high temperatures? In an elegant demonstration of how the Genomics Helps Explain Why Some Like It Hot DOI: 10.1371/journal.pbio.0030317 ever-growing publicly available genome sequence and protein structure data can be analyzed, Todd Yeates and colleagues identify one answer to this question.
The authors found that proteins from P. aerophilum and T. thermophilus, along with some other thermophiles, have many disulfi des, which are known to improve stability. Disulfi des are covalent bonds that form when the sulfhydryl groups (a sulfur and a hydrogen atom) of two spatially proximate cysteines (one of the 20 amino acid building blocks of proteins) are oxidized. When conditions are right, the two hydrogen atoms are removed by other molecules in the cell dedicated to that purpose, and the remaining sulfur atoms form a bond.
The authors mapped sequences of intracellular genes from 199 prokaryote genomes onto sequence-related proteins with known three-dimensional structures. The resulting structural models reveal when disulfi de bonds are likely to form. A pronounced bias was found for disulfi des in a set of thermophilic genomes. To prove that these predictions really do form disulfi de bonds, the authors solved the structure of one protein from P. aerophilum-which was indeed stabilized by three disulfi de bonds.
Disulfi de bonds form more commonly outside or between cells in multicellular organisms, where the environment is ideal for two cysteines to cozy up and bond in an oxidative extracellular location. The high numbers of bonds observed in these single-cell prokaryotes not only help explain thermostability but also challenge our ideas of how disulfi de bonds form. Given the presumed diffi culty for disulfi des to form in such organisms, the authors set out to look for any proteins that might help explain the mystery. They investigated which proteins are present in the disulfi de-rich organisms as compared with the proteins in other organisms (also known as phylogenetic profi ling). The authors discovered that all of the disulfi de-rich thermophiles had something else in common: they all encode a protein not seen in other organisms, called protein disulfi de oxidoreductase (PDO). As its name suggests, this protein likely plays a key role in the formation of disulfi des in these heat-tolerant bugs.
Yeates and colleagues have considerably advanced our understanding of how proteins withstand and remain functional at high temperatures in these thermophilic organisms (via additional stabilizing disulfi de bonds). Yet, since this correlation of extra disulfi des and the PDO is not common to all thermophiles, it seems likely that this is not the only method employed in heat resistance. Probably a fi nely tuned concert of different mechanisms works in synchrony to enable thermophiles to fl ourish in extreme conditions. As the authors show here, it's likely that genome sequence and structure data can help us to uncover these mechanisms. Have you ever wondered how the myriad insect forms-beetles, fl ies, dragonfl ies, mosquitoes, grasshoppers, ants, wasps, bees, and countless others-evolved? Insects make up 75% of all species known. The large number of insect species is probably a result of a combination of one or more factors: a high rate of formation of new species, or speciation, an ability to adapt to new environments and exploit new ecological niches, and a lower rate of extinction. Speciation, adaptation, and extinction are all controlled by the interplay between genetic and environmental factors. Understanding the genetic changes that lead to the formation of new species is an important area of research in evolutionary biology.
In a new study, Thomas Turner, Matthew Hahn, and Sergey Nuzhdin worked with the malaria mosquito Anopheles gambiae to uncover genes that may be driving speciation. A. gambiae exists in multiple forms that may be in the early stages of differentiating into separate species; on the other hand, they may be partially differentiated, co-existing races that could give us valuable information on genes responsible for racial differences in mosquitoes. Turner and colleagues focused on two forms, A. gambiae M and A. gambiae S, that sometimes mate and create hybrid forms in nature. While it's unclear whether the forms can produce fertile hybrid offspring in the wild, the progeny of lab matings appear to have no problems with fertility. This suggests that individuals either naturally prefer to mate with others of their own form, or that there must be environmental and/or genetic conditions that are not favorable for the survival of hybrid progeny in nature.
To study the genetic underpinnings of speciation, the researchers used DNA microarrays to identify global differences Islands in the Genome Promote Speciation in the mosquito genomes. Using a combination of gene chips, statistics, and computational biology, Turner and colleagues found that the M and S genomes differ at just three regions. The researchers suggested that genes present here may be responsible for early speciation. These three "speciation islands" in the genome contain 67 predicted genes. In a preliminary analysis of seven of these genes, Turner and colleagues identifi ed fi ve that are different between the two Anopheles forms; these include genes that play a role in a range of cellular processes, including energy metabolism, response to sudden increases in temperature (heat shock), and ion transport across cell membranes. Future work focusing on the 67 genes hypothesized to reside in the divergent regions should yield interesting clues to the identity of genes that drive speciation, and the mechanism by which they do so. This is a signifi cant fi nding in the fi eld of speciation research: in terms of methodology, this study shows that DNA microarrays can be used to identify regions of the genome that are different between two diverging species, allowing researchers to home in on potentially interesting genes. This study also shows that in spite of possible cross-fl ow of genetic material (natural hybrids between the two forms are found at a low frequency) between two populations, the populations can still be accumulating differences in their genomesdifferences that could eventually lead to the formation of new species. Comparing results in Anopheles and the well-studied insect model Drosophila, in which scientists have also started identifying "speciation genes," should tell us if similar genes are employed repeatedly in different genera during the formation of new species. The coil of a snail shell can be either right-handed (dextral) or left-handed (sinistral), based on whether the shell spirals out clockwise or counterclockwise when viewed from above. Most species are composed entirely of individuals that are one or the other type; in exceptional cases, populations may differ in their handedness, or chirality, but within a single population, all individuals tend to be alike. This makes sense, since the mechanics of reproduction are harder between two individuals of opposite chirality (their genitalia are also reversed), reducing the likelihood that they will successfully mate and produce offspring. Over time, therefore, the rarer type will become rarer and rarer until it goes extinct.
This poses the interesting evolutionary question of how a species of one chirality can give rise to another of opposite chirality. If the rarer types are less likely to reproduce, then how do they ever establish themselves beyond a threshold frequency? If they are able to establish themselves, then is a change in chiralitywhich is caused by a single geneenough to isolate them so that they are a new species? A study in this issue by Angus Davison et al. sheds light on the complex interplay of factors that infl uence evolution in the snail Euhadra. Although a single gene does cause a change in chirality, and snails with different chirality are able to mate only with great diffi culty, there is nevertheless almost free gene fl ow between them. Other factors must ultimately become involved to cause speciation.
The 22 species of Euhadra are landdwelling natives of Japan, and include fi ve sinistral and 17 dextral species. Using mitochondrial DNA analysis to construct a family tree, the authors showed that the sinistral species compose a distinct branch, indicating that this feature arose only once in the history of the genus. How did the fi rst sinistral shell types arise, and why didn't they gradually evaporate from the gene pool? One possibility is "reproductive character displacement," in which a new feature that directly affects mating, such as sinistral shell chirality, decreases the likelihood that its owner will mate with snails of other, closely related, species that live nearby. While their dextral brothers or sisters waste valuable resources in such unsuccessful interspecifi c pairings, the few sinistral individuals engage in fewer, but more productive, matings exclusively with their own kind, thus increasing their numbers despite the odds stacked against them.
To test this hypothesis, Davison et al. constructed a model that took into account a variety of factors, including population density, the proximity of other species, and the maternal inheritance pattern of shell chirality (the direction of a snail's shell is determined so early in development that it is governed not by its own genes, but by its mother's). The surprising conclusion is that the last factor, the unusual mode of inheritance, allows for near free gene fl ow between the two forms within a population, even if the two forms are themselves almost unable to mate. The reason is that the offspring of a sinistral mother could itself be sinistral, even if it contains entirely dextral genes. Its offspring, though, might include dextral snails, because its own dextral genes determined their shell chirality.
Their model indicated that new chiral types are able to arise, in spite of there being fewer suitable mates, if there is reproductive character displacement. They cannot be considered new species, however, because of the gene fl ow between them. Reproductive character displacement can account for the speciation of sinistral Euhadra only under a complex set of conditions. Interspecifi c mating would need to be common among the dextral snails. High population density helps, since it allows those with the rare new form to fi nd each other more easily. But gene fl ow between left and right forms would preserve the population as a single species, unless other factors, such as difference in habitat use or geographic separation, increased the isolation of the two forms. This argues against so-called "single-gene speciation," and shows that the creation of a new species requires more than a simple twist of fate. Last October, as Americans started lining up for fl u shots, news broke that 48 million vaccine doses had been contaminated. With 100 million people considered at high risk and fears of a potentially deadly avian fl u epidemic on the horizon, the shortage caused long lines, allegations of price gouging, and a new bill to bolster the nation's anemic vaccine manufacturing base.
Infl uenza A viruses are RNA viruses that infect humans, pigs, horses, and birds, both wild and domestic. Flu infection relies on a viral glycoprotein, hemagglutinin (HA), that binds to receptors on a host cell and allows the virus to be internalized. If antibodies produced by host immunity recognize viral antigens (on the surface of the HA protein), HA binding is inhibited and infection prevented. A virus's best chance of gaining the upper hand in this evolutionary game of cat and mouse is to change its HA in a way that eludes antibody recognition. Typically the mutations are minor and the virus's antigens conserved enough for the host body's immune system to recognize them. On occasion, infl uenza can acquire an antigenically novel HA subtype, becoming a virulent pandemic strain that completely escapes immune surveillance and kills millions. Minimizing the effect of yearly infl uenza outbreaks-by developing effective matched vaccines-depends on predicting which fl u strains are likely to evolve.
Toward this end, Eddie Holmes and colleagues took the global approach afforded by genomics to explore the forces underlying viral adaptations. In all the hullabaloo about stem cells, nobody has noted their uncanny similarity to pizza dough. You can divide either into two or four or eight identical pieces, but that doesn't determine what kind of cell or pizza you're going to make. But once you let a cell grow hundreds of nuclei, or you pile on the pepperoni, you're on your way to making a skeletal muscle fi ber or a pepperoni pizza. If you want a white blood cell or an all-veggie pie, you're out of luck. The commitment to becoming a certain cell type is called differentiation.
Stem cells in living organisms can multiply without differentiating, preserved by molecular signals in special niche environments; without these signals in the petri dish, they differentiate. Pluripotent mouse embryonic stem (ES) cells, a special type of stem cell with the potential to develop into many different cell types, are an exception. Because they divide symmetrically, the scads of artifi cially grown ES cells are all the same. This leads researchers to wonder: what conditions in the body keep stem cells from differentiating, why are ES cells the only kinds that don't differentiate in the petri dish, and how can scientists create undifferentiated tissue-specifi c stem cells in the lab?
In a new paper, Austin Smith and colleagues developed a method to produce symmetrical divisions of mouse brain stem cells derived from ES cells. Their novel method creates an on/off switch for differentiation of tissue-specifi c stem cells: they can multiply without differentiation, and they can also become normal brain cells. The authors also managed to cultivate the brain stem cells without re-creating the rarefi ed neurosphere, the highly specialized environment or microenvironment in which the body grows its own brain stem cells.
Many scientists believe that in the body, these microenvironments prevent stem cells from differentiating. Neurospheres, for example, contain some undifferentiated brain stem cells fl oating in a broth of differentiating cells. One feature of the neurosphere is that a very low percent of cells are brain stem cells. In fact, neurospheres have so few of these cells that scientists have a hard time even observing them. But by cultivating brain stem cells outside the neurosphere, the scientists showed that a complex microenvironment may not be necessary. To grow their stem cells, Smith et al. combined epidermal growth factor (EGF) and fi broblast growth factor (FGF), two small proteins that bind to stem cells and promote growth.
Previously, scientists had grown brain stem cells with FGF. Upon removing FGF, the cells failed to differentiate and become mature. The cells that Smith et al. grew, in contrast, became mature cells upon removal of the growth factor cocktail. They observed both neurons and astrocytes, the two types of cells into which the brain stem cells mature.
In the future, scientists may use this new technique to produce large quantities of the cells to study their basic properties and also to explore their value for modeling neurodegenerative affl ictions, including Huntington disease, Parkinson disease, and Alzheimer disease. Additionally, these cells may clinch the debate of whether doctors will be able to use stem cells directly to repair brain damage. Insulin or insulin-like proteins signal developing animals to grow. After a meal, the body creates insulin, allowing an organism to grow and compete with other organisms for available food. When food is scarce, insulin levels remain low. Only small organisms with low metabolic needs will survive the potential famine. Scientists can study how genes involved in insulin signaling affect development by mutating a gene and seeing what happens to the adult. This useful method, called gene knockout, provides insight into the specifi c relationship between a gene and its physical manifestation, or phenotype. By using the knockout method, scientists can observe how the growth of an organism responds to fl uctuations in insulin signaling levels.
In a new study, Alexander Shingleton and colleagues used a temperaturesensitive mutation in an insulin-receptor gene to discover how alterations of insulin signaling in the fruitfl y Drosophila affect different stages of fl y development. At one stage, the researchers discovered, insulin signaling infl uences total development time, at another it infl uences body size, and at a third stage, it infl uences only organ size.
So when do developing fl ies need insulin? The researchers found that low insulin signaling during very early development extends total development time. Then the larvae reach their critical size, the watershed moment in insect development when larvae commit to becoming pupae. After critical size, reduced insulin signaling no longer delays development but instead leads to petite fl ies with petite organs. When the larvae become pupae, however, reduced insulin signaling simply creates smaller organs. Because developmental time, body size, and organ size each display different responses to reduced insulin signaling activity, these features may evolve independently, the authors reasoned.
Shingleton and colleagues used fl ies with mutant insulin-receptor genes whose protein products are partially inactivated at high temperatures. circulating in the population at the same time, and a more complex evolutionary pattern than previously thought. They also showed that co-circulating viruses can exchange genes in a way that creates antigenically novel, epidemiologically signifi cant strains-a process that humans may facilitate by simultaneously hosting more than one strain.
They analyzed the genomes of 156 infl uenza A viruses (serotype H3N2) collected by New York State public health offi cials between 1999 and 2004 in search of global patterns of viral evolution. Using the fl u virus genome sequences produced at the Institute for Genomic Research (TIGR), funded by a National Institute for Allergy and Infectious Diseases (NIAID) initiative, the authors grouped the viral sequences according to sequence similarity. They also included partial fl u sequences obtained from other studies in their analysis. These data are the initial output of the fi rst large-scale effort to completely sequence infl uenza genomes. While most of the virus genomes sampled after 2002 fell into one group-which the authors called clade A-there were also other clades circulating at different times (called clades B and C).
Gene trees, or phylogenies, constructed for each of the virus's eight genes all diverged according to their respective clades, except one-the HA gene. The HA gene cluster grouped all the clade A viruses that emerged after 2002 as well as both the clade B and C viruses from the same time period and viruses from multiple locations (in Asia, Australia, Europe, and North America).
Altogether, these results indicate that different viral strains had circulated in the same populations until 2002 and then the clade A and C viruses acquired a common HA gene from clade B through reassortment. While reassortment between co-circulating human infl uenza strains has been previously described, this study is the fi rst to examine in detail a reassortment event leading to an epidemiologically signifi cant outcome, the emergence of the "Fujian" strain in the 2003-2004 season. Though it's not yet clear how variant clades manage to persist alongside dominant strains, the fact that they do suggests the infl uenza virus has multiple adaptive tools at its disposal. Luckily, the tools of genomics should help predict what evolutionary paths the virus might take and help in the process of selecting the most promising vaccines to contain it.

A transmission electron micrograph of the infl uenza A virus. New evidence suggests that fl u viruses can rapidly reshuffl e genetic material and mutate into new strains capable of widespread infection. (CDC/Dr. Erskine Palmer)
Malaria is a misnomer. People used to believe that poisoned or "bad air," the translation of the Italian phrase "mal aria," caused disease. In the 19th century, when parasitologists fi gured out that single-celled parasites cause malaria, they didn't bother to change the disease's name. Experimenters proved that these parasites need a host organism to survive-so they can't be transmitted through air-and that the hosts, mosquitoes, carry the parasite to humans. Researchers were optimistic that if they could fi nd a disease's cause, they could also fi nd the cure. Kill the mosquitoes and eradicate malaria. And with the advent of DDT and less environmentally harmful insecticides, potent anti-malarial drugs, and international funding in the late 20th century, eradication of malaria seemed imminent.
But that expectation underestimated the fl exibility of living creatures. Mosquitoes acquired resistance to insecticides while the parasites acquired resistance to anti-malarial drugs. Worse, the aggressive eradication campaign skipped over vast regions of the globe, especially sub-Saharan Africa.
Malaria remains a devastating problem in Africa for several reasons. Environmental conditions provide an amenable atmosphere for both Plasmodium falciparum, the most dangerous form of the parasite, and the Anopheles gambiae mosquito, the most effective vector. Also, many countries in sub-Saharan Africa lack the infrastructure to protect their citizens from malaria. Given the overwhelming scope of malarial infection in Africa, new understanding of the disease will help epidemiologists devise targeted antimalarial strategies.
A new study conducted in Western Kenya by Jacob Koella and colleagues analyzed mosquito behavior to discover how it facilitates the transmission of malaria. The research determined that mosquitoes are more attracted to people infected with transmittable malaria than to either people infected with non-transmittable forms of the disease or uninfected people. To measure the attraction of the mosquitoes, the researchers set up a chamber of infected mosquitoes surrounded by tents containing the study participants. A device called an olfactometer wafted the odors of each participant toward the mosquitoes. Researchers measured which smell most attracted the hungry bugs.
This question had long stalled scientists because of contradictory and indirect evidence. Sweat, breath odor, and high body temperature all increase mosquitoes' blood lust, and no previous study had isolated the variable of malarial infection.
To control for the natural variation in how attractive mosquitoes found each participant, Koella et al. compared the number of mosquitoes that were attracted to infected people to the number of mosquitoes that were attracted to those same people after they were no longer infected. The researchers found that in general, an individual attracted more mosquitoes when infected with transmittable malaria. This demonstrates that malaria, in addition to causing fever, vomiting, headache, and sometimes death, causes more mosquito bites. The biting mosquitoes will then pick up the parasite and spread it to other people.
As another control, the researchers compared infection with a nontransmittable form of the parasite to infection with the transmittable form and to no infection. A mosquito can pick up the malaria parasite only when in its sexually reproductive stage. The transmittable parasite, known as a gametocyte, multiplies in the mosquito's belly before traveling to the mosquito's salivary glands and, eventually, to the blood of the next human victim. But the malaria parasite has a complicated life cycle that also includes non-transmittable asexual stages. Koella and colleagues found that these parasitic forms, unlike the sexually reproductive form, did not make humans more attractive to mosquitoes.
Previous to the recent study, malaria researchers had proved that mosquito biting rates greatly infl uence the spread of malaria. Koella and colleagues showed that the parasite itself increases these biting rates when it is ready for a new host. watched what happened to their bugs. Using this temperature-sensitive insulinreceptor gene, the researchers found that, besides affecting development time, insulin signaling also plays a role in the differential growth rates of different organs. By tracking three organs on male fl ies, Shingleton and colleagues discovered that the genitals are less sensitive to reduced insulin signaling than either the wings or the maxillary palps, olfactory components of the mouth. The authors also found that insulin signaling affects cell size and cell number differently. While slightly reduced insulin signaling shrinks cell size, highly reduced insulin signaling lowers cell number without affecting cell size. By incorporating the effects of reduced insulin signaling into the Drosophila development process, the authors constructed a model of Drosophila development that explains the various roles played by the insulin-signaling pathway during development.
Because the new study alters genes during development, it provides the details of when and how a developing animal requires insulin. Future fl y studies may reveal why organs have individual responses to insulin signals, what other signaling pathways play a role in development, and how insulin came to infl uence so many different features of the developing fl y at different times. To control what you eat and when, your nervous system must coordinate a laundry list of signals: internal signals contain information about energy level, food preferences, and metabolic need, while external signals relay information about the quality of available food, determined by its smell and taste. Scientists studying the fruitfl y Drosophila have traced the path of olfactory signals beginning with chemical receptors in the mouth, which set off neurons that signal the antennal lobe of the central nervous system. From here, the electrical stimulation zooms toward the so-called mushroom body, a mushroom-shaped cluster of neurons involved in olfactory processing. Less is known about the gustatory signals, which begin both in the mouth and in the pharynx and aim toward the subesophageal ganglion region of the fl y's brain. How olfactory and gustatory signals infl uence feeding patterns remains murky.
In a new study, Michael Pankratz and Christoph Melcher used genetic analysis to gain insight into the adult and larval neural networks that use taste information to regulate eating. Specifi cally, they found that several types of neurons responsible for coordinating taste signals express the gene hugin (hug), a gene linked to abnormal eating activity and expressed in only the subesophageal ganglion. By altering hug expression, the researchers uncovered the gene's behavioral infl uence: hug-expressing neurons infl uence a fl y's decision to sample new food sources. The researchers also proposed that hug proteins play a role in hormone-triggered growth, an important consequence of adequate feeding.
To begin their investigation, Melcher and Pankratz analyzed the DNA from fl ies with abnormal eating behavior. One group of these fl ies shared a mutant klumpfuss (klu) gene, normally responsible for encoding a protein transcription factor. Because neural transcription factors control production levels of other neural proteins, the researchers used DNA microarrays to compare gene expression in normal fl ies to that in klu mutants. Any klu-controlled genes expressed at different levels in klu mutants might contain clues about the neural circuitry modulating feeding behavior.
Using microarrays, Melcher and Pankratz discovered that mutant fl y larvae overexpress the hug gene, which is known to encode at least two neural proteins related to growth signaling. The researchers then investigated which signals infl uence hug expression by exposing larvae to either high or low food levels. Because both starved and sugar-fed fl ies express little hug, the researchers inferred that hug levels do not solely signal internal energy requirements but respond to internal and external signals carrying information about the quality of food. The researchers also noted that the fi nicky pumpless (ppl) mutants, which have a feeding defect similar to klu, overexpress hug.
Behavioral studies confi rmed that too much hug reduces food intake and leads to stunted growth, while too little stimulates eating. Melcher and Pankratz selected a group of fl ies and blocked the synapses of their hug neurons to inhibit the neurons' activity. In contrast to control fl ies, which start feeding on a novel food source only after an evaluation phase (they wait a while before initiating feeding), the experimental fl ies started eating new food right away. These hug neurons may help fl ies decide whether or not to eat a new food source.
Larvae express hug in only about 20 neurons, all located in the subesophageal ganglion. The axons of some of these hug neurons extend into the ring gland, a crucial metabolism and growth organ in fl ies. Other axons contact the protocerebrum, a structure close to brain centers for learning and remembering odors. A third set of these axons extend to throat muscleswhich is surprising because most subesophageal ganglion neurons have no connection to motor function. All together, these few hug neurons can signal structures controlling growth, feeding, and learning and memory.
Besides linking hug neurons to brain centers that regulate taste-related feeding behavior, the study also raises questions about how the nervous system prioritizes internal and external signals. How hungry must fl ies be to overcome taste aversion? How do the competing neural networks of taste and hunger signals decide whether the fl y will eat? Future studies pairing behavioral and genetic analysis may begin to reveal answers to these open questions. If you snooze, you lose those uncomely grayish-brown crescents below your eyes. If you don't snooze, you lose a lot more. The body can't fi ght off infection, the muscles can't regenerate as quickly, the mind can't learn new words, and the eyes can't focus on the road. You also gain things: a bad mood and increased risk for diabetes, high blood pressure, and heart problems. Indeed, the effects of sleep deprivation can be so serious that some sleep scientists liken lifetime sleep debt to a heavy backpack: every sleep hour missed adds an extra pound to your pack until it weighs you down.
For people without time for a daily eight hours in the sack, drugs that counteract the effects of sleep deprivation could serve as substitutes. In a new study, Sam Deadwyler and colleagues have explored this possibility by giving dog-tired rhesus monkeys a drug shown to improve the functioning of alert brains. They found that sleepy monkeys taking the drug performed tasks better and had increased metabolic activity in several regions of their brains. This suggests that the cognitive effects of sleep deprivation can be reduced chemically.
The researchers kept the monkeys awake for 30 to 36 hours by playing music and videos, keeping the lights on, and interacting with them: all the annoyances that can also keep humans from sleeping. To determine the drug's effect on drowsy monkeys, Deadwyler and colleagues used a behavioral test called Match-To-Sample, which measured both accuracy of memory and speed of recall. In the behavioral test, the monkeys saw a simple image fl ash on a screen. For a variable amount of time, the monkeys had to remember the image. Then, they had to select the correct image from a group of others shown on the monitor simultaneously. When monkeys correctly selected the original image with a cursor, they got a squirt of juice in their mouth as reward. The researchers measured how long they could keep the screen blank between the fi rst and second images without affecting the monkeys' performance. They found that if the monkeys were tired, they couldn't remember the fi rst image for long as they could when they were alert. But with the drug, the sleepdeprived monkeys did at least as well as alert monkeys.
The drug, labeled CX717 (Cortex Pharmaceuticals), acts on AMPA receptors, protein structures on the surface of neurons. When these receptors bind to the neurotransmitter glutamate, they transduce excitatory signals by opening an ion channel. Ampakines including CX717 make the activated channel stay open longer when glutamate binds. More ions pass through the channel, creating a stronger signal when nerve cells are activated. The ubiquity of these receptors makes them good targets for drugs that increase general cognitive functioning. The researchers used a technique called positron emission tomography, or PET, to gain insight into CX717's neurobiological role. The PET signal refl ected the distribution and rate of metabolism of ingested radioactively labeled glucose in the monkeys' brain cells. By measuring regional brain glucose metabolism, the researchers determined that for sleepdeprived monkeys, glucose metabolism drops off in brain areas previously associated with memory tasks-namely, the prefrontal cortex, the dorsal striatum, and the medial temporal lobe. However, when sleep-deprived monkeys took the drug, they showed heightened glucose metabolism in these same brain regions. The researchers compared these results to suggest a biological basis for the drug's effects.
Previous studies have shown that caffeine and amphetamine can reduce the deleterious cognitive effects of sleep deprivation. But as anybody who has indulged one latte too many knows, caffeine and other powerful stimulants have limited usefulness. These potentially addictive chemicals can distort thinking just as they can enhance it. Because CX717 has a different biochemical action, it may be more benefi cial than stimulants for counteracting the cognitive effects of sleep deprivation. But that doesn't mean we should throw away our pillows and blankets just yet: sleep deprivation affects both body and mind. Obesity is a leading cause of preventable death and is often linked to type II diabetes and heart disease. Being a complex trait, obesity is likely caused by the interplay of multiple environmental factors and many genes. Common genetic differences between individuals within a region of Chromosome 10 have previously been associated with obesity. This region contains several genes with the potential to be directly involved in the disease. One of these genes, GAD2, has been the subject of many studies. A new study by Michael Swarbrick, Björn Waldenmaier, Christian Vaisse, and their colleagues takes a new look at GAD2 and provides strong evidence that the gene might not be as relevant to obesity as previously thought.
GAD2 encodes a protein (called GAD-65) involved in the production of GABA, a neurotransmitter involved in a variety of brain functions, including appetite stimulation and energy consumption. Studies in mice have shown that increased levels of GABA result in hunger and overeating. In healthy mice, the levels of GAD2, and hence, GABA, are controlled, making sure that the balance between weight gain and loss is maintained. A 2003 study of a French population found that three genetic mutations in and around the GAD2 gene occurred at a high level in individuals with obesity. The 2003 study, conducted by different researchers, A Genetic Link to Obesity: The Numbers Don't Add Up for GAD2 DOI: 10.1371/journal.pbio.0030321 was also published in PLoS Biology. When Swarbrick et al. surveyed German, Caucasian-American, and Canadian populations for this genetic correlation, however, they found no statistically signifi cant link between obesity and any of the mutations.
There are many possible reasons why different studies may show different results: ethnic differences between populations, as well as behavioral and dietary differences, could account for varying results when it comes to studying a trait as complex as obesity. Also, studies that seek to show an association between genetic differences and complex diseases rely heavily on the statistical power of their tests, which depends on the number of subjects involved. Swarbrick et al. have not only studied 2,359 German, 729 US, and 1,137 Canadian subjects, but also conducted a "meta-analysis"-a statistical analysis of a collection of individual studies-of their data and the previously published data from 1,221 French subjects. Metaanalyses help identify patterns from multiple individual studies that may not be visible in any one study alone, and also help rule out chance differences that may be apparent in one single study. In this case, the meta-analysis showed that when the results from French subjects are put together with the results from other ethnic populations, there is no evidence for a link between changes in GAD2 and obesity.
Although GAD2's role in controlling appetite made it an exciting candidate for a link to obesity-related conditions, Swarbrick et al. show that the numbers simply don't add up. The search for serious obesity gene contenders in this region of Chromosome 10 is all set to continue-and attention can now turn to several other potential gene candidates located nearby. No matter how healthy a life one leads, no person has managed to live much longer than a century. Even though the advances of the modern age may have extended the average human life span, it is clear there are genetic limits to longevity. One prominent theory of aging lays the blame on the accumulation of damage done to DNA and proteins by "free radicals," highly reactive molecules produced by the metabolic activity of mitochondria. This damage is expected to reduce gene expression by damaging the DNA in which genes are encoded, and so the theory predicts that the most metabolically active tissues should show the greatest age-related reduction in gene expression. In this issue, Michael Eisen and colleagues show that the human brain follows this pattern. A similar pattern-which, surprisingly, involves different genes-is found in the brain of the aging chimpanzee.
The authors compared results from three separate studies of age-related gene expression, each done on the same type of DNA microarray and each comparing brain regions in young versus old adult humans. In four different regions of the cortex (the brain region responsible for higher functions such as thinking), they found a similar pattern of age-related change, characterized by changes in expression of hundreds of genes. In contrast, expression in one non-cortical region, the cerebellum (whose principal functions include movement), was largely unchanged with age. In addition to confi rming a prediction of the free-radical theory of aging (namely, that the more metabolically active cortex should have a greater reduction in gene activity), this is the fi rst demonstration that age-related gene expression patterns can differ in different cells of a single organism.
The authors found a similar difference in age-related patterns in the brain of the chimpanzee, with many genes down-regulated in the cortex that remained unchanged in the cerebellum. However, the set of affected cortical genes was entirely different between humans and chimps, whose lineages diverged about 5 million years ago. The explanation for this difference is unknown, but the fi nding highlights the fact that signifi cant changes in gene expression patterns, and thus changes in many effects of the aging process, can accumulate over relatively short stretches of evolutionary time.
These results raise a number of questions about age-related gene expression changes, including whether metabolically active non-brain tissues display similar patterns of changes, and whether the divergence between human and chimp patterns was the direct result of selection, or was an inevitable consequence of some other difference in brain evolution. The patterns seen in this study also provide a starting point for understanding the network of genetic changes in aging, and may even reveal targets for treatment of neurodegenerative diseases. Eyewitness testimony has a unique ability to convince juries. The attorney asks the witness to identify the guilty party. The witness points to the defendant, the crowd gasps, and the judge pounds her gavel, demanding order in the court. The jurors casually scribble something in their notes, and everybody knows that the fate of the accused has been sealed. But how reliable is a witness's memory, especially after rehearsing the testimony ad nauseam with a team of lawyers? When a witness presents testimony, is she really remembering the event, or is she remembering something she remembered? Does the initial memory remain intact, or does it degrade like a copy of a copy?
The status of witness testimony in court is just one reason neuroscientists want to understand the biochemical underpinnings of memory formation. Consolidation, the process of new memory formation that takes place in the hippocampus, requires certain proteins. Reconsolidation, the reactivation of these memories in the amygdala, requires a different set of proteins. In the past, neuroscientists hypothesized that reconsolidation might allow old and new memories to link up. A new study by Cristina Alberini and colleagues provides evidence that when rats link new memories to old, the molecular basis of this process actually resembles consolidation.
To manipulate lab rat memories, the researchers used constructions called inhibitory avoidance apparatuses. The fi rst apparatus had two tiny rooms: a well-lit safe room and a pitch-black electric-shock room. Rats spent ten seconds in the fi rst room, the researchers fl ipped on a light, and the rats entered the shock chamber.
Alberini and colleagues knew that the rats had formed a new memory when they hesitated to enter the dark room.
Rats then entered a second apparatus decorated differently from the fi rst apparatus. The safe room smelled of perfume, the walls displayed striped wallpaper, and the fl oor was made from smooth plastic. For rats in the second apparatus, the researchers fl ipped on a light but did not let the rats pass into the shock room. Alberini and colleagues deduced that the rats had compiled their memories of both the fi rst and second apparatuses when they hesitated to enter the second dark room during a fi nal test.
The researchers found that rats injected with anisomycin, a drug that inhibits protein synthesis, could not form a new memory of the second apparatus and sometimes forgot the fi rst. This showed that, as predicted, both the formation of new memories and the reconsolidation of old memories require protein synthesis. The researchers demonstrated the distinction between the processes of consolidation and reconsolidation by showing that rats require a certain protein in the hippocampus only for memory consolidation and the same protein in the amygdala only for reconsolidation.
Using a combination of proteins that took advantage of the differences between consolidation and reconsolidation, the researchers inhibited either the rats' consolidation mechanism or the reconsolidation mechanism.
Then, Alberini and colleagues tested the rats' ability to link their memory of the fi rst apparatus to their exposure to the second. Upon repeated trials, the rats with blocked reconsolidation pathways successfully linked memories of both apparatuses, while the rats with blocked consolidation pathways did not. Therefore, the consolidation pathway, and not the reconsolidation pathway, plays a role in memory linkage.
As a cautionary word, the researchers emphasized that their results applied to the fear-based memories created by the electric shock. Future studies may reveal if other types of memory yield the same results.