CO2 Capture in Ionic Liquids Based on Amino Acid Anions With Protic Side Chains: a Computational Assessment of Kinetically Efficient Reaction Mechanisms.

Absorption and capture of CO2 directly from sources represents one of the major tools to reduce its emission in the troposphere. One of the possibilities is to incorporate CO2 inside a liquid exploiting its propensity to react with amino groups to yield carbamic acid or carbamates. A particular class of ionic liquids, based on amino acids, appear to represent a possible efficient medium for CO2 capture because, at difference with current industrial setups, they have the appeal of a biocompatible and environmentally benign solution. We have investigated, by means of highly accurate computations, the feasibility of the reaction that incorporates CO2 in an amino acid anion with a protic side chain and ultimately transforms it into a carbamate derivative. Through an extensive exploration of the possible reaction mechanisms, we have found that different prototypes of amino acid anions present barrierless reaction mechanisms toward CO2 absorption.

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue.

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.

Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit.

Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.

Study on the clinical application of the MRS in the cognitive assessment after stroke.

OBJECTIVE: To discuss the value of the application of the magnetic resonance spectroscopy (MRS) in the assessment of cognitive function impairment and the observation of therapeutic effect. PATIENTS AND METHODS: 30 patients with cognitive impairment after stroke (cognitive impairment group) and 30 patients with no apparent cognitive impairment (control group of the stroke) were selected through the screening of the mini-mental state examination (MMSE) scale, and another 30 healthy volunteers were selected (control group of the health). The general information of gender, age, education degree, disease duration and so on was matched among the 3 groups. MRS examined all the patients of the 3 groups at the time of enrollment, and the patients of both cognitive impairment and control groups received another assessment 2 months after treatment. RESULTS: The NAA/Cr (N-acetyl aspartate/creatine) of the bilateral hippocampus of the cognitive impairment group was lower than those of both control group of stroke and health (p < 0.05), while the Cho/Cr was higher (p < 0.05). After treatment, both patients of the cognitive impairment group and the control group of the stroke experienced an increase of NAA/Cr and a decrease of Cho/Cr (p < 0.05). CONCLUSIONS: MRS is applicative in the assessment of the cognitive impairment degree of the stroke patients and can also effectively identify the existence of the cognitive impairment, which makes it preferably valuable in the clinical application.

Extensive Assessment of Various Computational Methods for Aspartate's pKa Shift.

A series of computational methods for pKa shift prediction are extensively tested on a set of benchmark protein systems, aiming at identifying pitfalls and evaluating their performance on high variants. Including 19 ASP residues in 10 protein systems, the benchmark set consists of both residues with highly shifted pKa values as well as those varying little from the reference value, with an experimental RMS free energy differences of 2.49 kcal/mol with respect to blocked amino acid, namely the RMS pKa shift being 1.82 pKa units. The constant pH molecular dynamics (MD), alchemical methods, PROPKA3.1, and multiconformation continuum electrostatics give RMSDs of 1.52, 2.58, 1.37, and 3.52 pKa units, respectively, on the benchmark set. The empirical scoring method is the most accurate one with extremely low computational cost, and the pH-dependent model is also able to provide accurate results, while the accuracy of MD sampling incorporating alchemical free energy simulation is prohibited by convergence achievement and the performance of conformational search incorporating multiconformation continuum electrostatics is bad. Former research works did not define statistical uncertainty with care and yielded the questionable conclusion that alchemical methods perform well in most benchmarks. In this work the traditional alchemical methods are thoroughly tested for high variants. We also performed the first application of nonequilibrium alchemical methods to the pKa cases.

Production, characterization, and assessment of a stable analog of the response regulator CheY-phosphate from Thermotoga maritima.

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of ∼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.

Simulating the function of sodium/proton antiporters.

The molecular basis of the function of transporters is a problem of significant importance, and the emerging structural information has not yet been converted to a full understanding of the corresponding function. This work explores the molecular origin of the function of the bacterial Na+/H+ antiporter NhaA by evaluating the energetics of the Na+ and H+ movement and then using the resulting landscape in Monte Carlo simulations that examine two transport models and explore which model can reproduce the relevant experimental results. The simulations reproduce the observed transport features by a relatively simple model that relates the protein structure to its transporting function. Focusing on the two key aspartic acid residues of NhaA, D163 and D164, shows that the fully charged state acts as an Na+ trap and that the fully protonated one poses an energetic barrier that blocks the transport of Na+. By alternating between the former and latter states, mediated by the partially protonated protein, protons, and Na+ can be exchanged across the membrane at 2:1 stoichiometry. Our study provides a numerical validation of the need of large conformational changes for effective transport. Furthermore, we also yield a reasonable explanation for the observation that some mammalian transporters have 1:1 stoichiometry. The present coarse-grained model can provide a general way for exploring the function of transporters on a molecular level.

A comprehensive molecular dynamics approach to protein retention modeling in ion exchange chromatography.

In downstream processing, the underlying adsorption mechanism of biomolecules to adsorbent material are still subject of extensive research. One approach to more mechanistic understanding is simulating this adsorption process and hereby the possibility to identify the parameters with strongest impact. So far this method was applied with all-atom molecular dynamics simulations of two model proteins on one cation exchanger. In this work we developed a molecular dynamics tool to simulate protein-adsorber interaction for various proteins on an anion exchanger and ran gradient elution experiments to relate the simulation results to experimental data. We were able to show that simulation results yield similar results as experimental data regarding retention behavior as well as binding orientation. We could identify arginines in case of cation exchangers and aspartic acids in case of anion exchangers as major contributors to binding.

Investigation of asparagine deamidation in a SOD1-based biosynthetic human insulin precursor by MALDI-TOF mass spectrometry.

A biosynthetic human insulin precursor displayed enhanced susceptibility to deamidation at one particular site. The present study was undertaken to monitor progress of precursor deamidation at successive manufacturing stages. MALDI-TOF/TOF MS in combination with controlled endoproteinase Glu-C and endoproteinase Asp-N proteolysis was used for rapid and unambiguous determination of deamidated residue within the investigated structure. Close inspection of isotopic distribution patterns of peptides resulting from enzymatic digestion enabled determination of distinct precursor forms occurring during the production process. Asn, Asp, isoAsp and succinimide derivatives of the amino acid at position 26 were unambiguously identified. These modifications are related to the leader peptide of a precursor encompassing amino acid sequence corresponding to that of superoxide dismutase [Cu-Zn] (SOD1 1, EC=1.15.1.1). Monitoring of precursor deamidation process at successive manufacturing stages revealed that the protein folding stage was sufficient for a prominent replacement of asparagine by aspartic and isoaspartic acid and the deamidated human insulin precursor constituted the main manufactured product. Conversion proceeded through a succinimide intermediate. Significant deamidation is associated with the presence of SNG motif and confirms results achieved previously on model peptides. Our findings highlight an essential role of the specific amino acid sequence on accelerated rate of protein deamidation. To our knowledge, this is the first time that such a dramatic change in the relative abundance of Asp and isoAsp resulting from protein deamidation process is reported.

Effects of Zn2+ binding on the structural and dynamic properties of amyloid ß peptide associated with Alzheimer's disease: Asp1 or Glu11?

Extensive experimental and computational studies have suggested that multiple Zn(2+) binding modes in amyloid ß (Aß) peptides could exist simultaneously. However, consistent results have not been obtained for the effects of Zn(2+) binding on Aß structure, dynamics, and kinetics in particular. Some key questions such as why it is so difficult to distinguish the polymorphic states of metal ions binding to Aß and what the underlying rationale is, necessitate elucidation. In this work, two 3N1O Zn(2+) binding modes were constructed with three histidines (His(6), His(13), and His(14)), and Asp(1)/Glu(11) of Aß40 coordinated to Zn(2+). Results from molecular dynamics simulations reveal that the conformational ensembles of different Zn(2+)-Aß40 complexes are nonoverlapping. The formation of turn structure and, especially, the salt bridge between Glu(22)/Asp(23) and Lys(28) is dependent on specific Zn(2+) binding mode. Agreement with available NMR observations of secondary and tertiary structures could be better achieved if the two simulation results are considered together. The free energy landscape constructed by combining both conformations of Aß40 indicates that transitions between distinct Aß40 conformations thar are ready for Zn(2+) binding could be possible in aqueous solution. Markov state model analyses reveal the complex network of conformational space of Aß40 modeulated by Zn(2+) binding, suggesting various misfolding pathways. The binding free energies evaluated using a combination of quantum mechanics calculations and the MM/3D-RISM method suggest that Glu(11) is the preferred oxygen ligand of Zn(2+). However, such preference is dependent on the relative populations of different conformations with specific Zn(2+) binding modes, and therefore could be shifted when experimental or simulation conditions are altered.

An economical method for production of (2)H, (13)CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome.

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13)CH(3) methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2)H; ß-(2)H;γ-(13)C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr (13)C,(1)H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[(13)C,(1)H]-Thr.

Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures.

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

Conformation of a coarse-grained protein chain (an aspartic acid protease) model in effective solvent by a bond-fluctuating Monte Carlo simulation.

In a coarse-grained description of a protein chain, all of the 20 amino acid residues can be broadly divided into three groups: Hydrophobic (H) , polar (P) , and electrostatic (E) . A protein can be described by nodes tethered in a chain with a node representing an amino acid group. Aspartic acid protease consists of 99 residues in a well-defined sequence of H , P , and E nodes tethered together by fluctuating bonds. The protein chain is placed on a cubic lattice where empty lattice sites constitute an effective solvent medium. The amino groups (nodes) interact with the solvent (S) sites with appropriate attractive (PS) and repulsive (HS) interactions with the solvent and execute their stochastic movement with the Metropolis algorithm. Variations of the root mean square displacements of the center of mass and that of its center node of the protease chain and its gyration radius with the time steps are examined for different solvent strength. The structure of the protease swells on increasing the solvent interaction strength which tends to enhance the relaxation time to reach the diffusive behavior of the chain. Equilibrium radius of gyration increases linearly on increasing the solvent strength: A slow rate of increase in weak solvent regime is followed by a faster swelling in stronger solvent. Variation of the gyration radius with the time steps suggests that the protein chain moves via contraction and expansion in a somewhat quasiperiodic pattern particularly in strong solvent.

Oral colon targeted delivery systems for treatment of inflammatory bowel diseases: synthesis, in vitro and in vivo assessment.

The aim of this study was to investigate the potential of prodrugs of some non-steroidal anti-inflammatory drugs (NSAIDs) as colon targeted delivery systems for treatment of inflammatory bowel diseases. Naproxen, sulindac and flurbiprofen (Fbp) were used. The carboxylic group of those drugs was conjugated onto the amino group of l-aspartic acid or the hydroxyl group of alpha- or beta-cyclodextrin (CyD). Prodrugs hydrolysis in buffers of pH range 1.2-7.2 and in rat gastrointestinal tract homogenates and the effect of oral pretreatment of rats with clindamycin on the hydrolysis of the prodrugs was examined. Additionally, the effect of oral administration of Fbp-beta-CyD prodrug on the experimentally induced colitis in rats was evaluated. The in vivo inflammatory response was assessed macroscopically, histologically and by measurement of reduced glutathione (GSH) levels in colon tissues. No significant hydrolysis of the proposed seven prodrugs in buffers having pH range of 1.2-7.2 was observed over 72h. Negligible % of drug released from Fbp-alpha-CyD or Fbp-beta-CyD prodrugs was detected in rat stomach contents, intestinal tissues and intestinal contents homogenates. On the other hand, Fbp-alpha-CyD and Fbp-beta-CyD prodrugs released about 60% Fbp within 4h in rat colon homogenate. Oral pretreatment of rats with clindamycin significantly reduced % Fbp released from Fbp-alpha-CyD or Fbp-beta-CyD prodrugs. Oral administration of Fbp-beta-CyD to rats after induction of colitis significantly attenuated the severity of the colonic injury and reduced the score of the macroscopic and microscopic damage. Additionally, there was a significant increase in the level of GSH. The present study provided an evidence that Fbp-beta-CyD prodrug may be beneficial in treatment of inflammatory bowel disease.

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

The aim of the present study was to identify specific alpha(v)beta3/alpha(v)beta5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp-containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for alpha(v)beta3 and alpha(v)beta5 integrins with negligible interacting with alpha5beta1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of alpha(v)beta3/alpha(v)beta5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of alpha(v)beta3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-alpha(v)beta3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-alpha(v)beta3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual alpha(v)beta3/alpha(v)beta5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Analysis and prediction of leucine-rich nuclear export signals.

We present a thorough analysis of nuclear export signals and a prediction server, which we have made publicly available. The machine learning prediction method is a significant improvement over the generally used consensus patterns. Nuclear export signals (NESs) are extremely important regulators of the subcellular location of proteins. This regulation has an impact on transcription and other nuclear processes, which are fundamental to the viability of the cell. NESs are studied in relation to cancer, the cell cycle, cell differentiation and other important aspects of molecular biology. Our conclusion from this analysis is that the most important properties of NESs are accessibility and flexibility allowing relevant proteins to interact with the signal. Furthermore, we show that not only the known hydrophobic residues are important in defining a nuclear export signals. We employ both neural networks and hidden Markov models in the prediction algorithm and verify the method on the most recently discovered NESs. The NES predictor (NetNES) is made available for general use at http://www.cbs.dtu.dk/.

The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion.

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.

Conformational analysis of the biologically active RGD-containing anti-adhesive peptide cyclo(ArgGlyAspPhe-D-val).

Using theoretical conformational analysis, the RGD-peptide with anti-adhesive activity cyclo(ArgGlyAspPhe-D-Val) was studied. Random sampling was used to search the conformational space of the allowed torsional angles of the main chain of the molecule. Among 900 stable conformers with different folding of the cyclic moiety of the peptide, only those were selected which corresponded to low-energy conformers of the model linear tripeptide AcAlaGlyAspNHMe. This peptide served as the main chain template of the RGD-fragment of the studied cyclopeptide molecule. Of 36 selected cyclopeptide conformers with potential biological activity, only a few contain stable intramolecular hydrogen bonds. It was supposed that a biologically active conformer of the cyclopeptide molecule exists in solution among other conformers, but not necessarily as the major component of the equilibrium mixture.

Haemochromatosis: automated detection of the two point mutations in the HFE gene: Cys282Tyr and His63Asp.

Hereditary haemochromatosis (HH) is one of the most common inherited diseases among Caucasians. Two mutations in the HFE gene have been implicated in HH: 80 to 90% of the patients with HH are homozygous for the point mutation CYS282Tyr, while the majority of the remaining patients displays either a compound heterozygosity for the mutation CYS282Tyr and the point mutation HIS63Asp, or are homozygous for HIS63Asp. Though the disease can be treated easily, symptoms are non-specific, and onset and severity are influenced by environmental factors, and therefore the disease can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, which may result in their failure. In order to detect patients with HH, simple and cost-effective tests are needed. We have developed a rapid, automated, PCR-based test which makes use of a diagnostic restriction site in each of two amplified fragments. The test employs off-the-shelf chemistry and uses the automated detection process of an immunoassay analyzer that is available in many clinical laboratories, thus avoiding an additional investment in a more specialized PCR analyzer. Because of its low costs and easy handling, the assay is particularly suited for the routine clinical laboratories.

Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit.

The lack of knowledge of the three-dimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-A crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analog-bound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The flexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.