Design, synthesis and evaluation of transition-state analogue inhibitors of Escherichia coli gamma-glutamylcysteine synthetase

Phosphinic acid-, sulfoximine- and sulfone-based transition-state analogues were synthesized and evaluated as inhibitors of Escherichia coli gamma-glutamylcysteine synthetase. These compounds have a carboxyl function at the beta-carbon to the tetrahedral central hetero atom so as to mimic the carboxyl group of the attacking cysteine in the transition state. The phosphinic acid- and the sulfoximine-based compounds were found to be potent ATP-dependent inactivators, both showing a slow-binding kinetics with overall affinities and second-order inactivation rates of one to two orders of magnitude greater than those of L-buthionine (SR)-sulfoximine (L-BSO). The sulfone was a simple reversible inhibitor without causing ATP-dependent enzyme inactivation, but its affinity toward the enzyme was still five times greater than that of L-BSO, indicating that the beta-carboxyl function plays a key role in the recognition of the inhibitors by the enzyme. The sulfoximine with (S)-beta-carbon to the sulfur was synthesized stereoselectively, and the two diastereomers with respect to the chiral sulfur atom were separated as a cyclic sulfoximine derivative. The sulfoximine with R-configuration around the sulfur served as an extremely powerful ATP-dependent inactivator with an overall inhibition constant of 39 nM and an inactivation rate of 6750 M-1 s-1, which correspond to 1260-fold higher affinity and almost 1400-fold greater inactivation rate as compared with L-BSO. The sulfoximine with (S)-sulfur was a simple reversible inhibitor with an inhibition potency comparable to that of the sulfone. The synthesis and inhibition profile of the N-phosphoryl sulfoximine is also described.     

Three-dimensional structure of Escherichia coli dihydrodipicolinate reductase in complex with NADH and the inhibitor 2,6-pyridinedicarboxylate

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the biosynthetic pathway that leads to diaminopimelate and lysine in bacteria and higher plants. Because these pathways are unique to microorganisms and plants, they may represent attractive targets for new antimicrobial or herbicidal compounds. The three-dimensional structure of the ternary complex of Escherichia coli dihydrodipicolinate reductase with NADH and the inhibitor 2,6-pyridinedicarboxylate has been solved using a combination of molecular replacement and noncrystallographic symmetry averaging procedures and refined against 2.6 A resolution data to a crystallographic R-factor of 21.4% (Rfree is 29.7%). The native enzyme is a 120 000 molecular weight tetramer of identical subunits. The refined crystallographic model contains a tetramer, three molecules of NADH, three molecules of inhibitor, one phosphate ion, and 186 water molecules per asymmetric unit. Each subunit consists of two domains connected by two flexible hinge regions. While three of the four subunits of the tetramer have a closed conformation, in which the nicotinamide ring of the cofactor bound to the N-terminal domain and the reducible carbon of the substrate bound to the substrate binding domain are about 3.5 A away, the fourth subunit is unliganded and shows an open conformation, suggesting that the enzyme undergoes a major conformational change upon binding of both substrates. The residues involved in binding of the inhibitor and the residues involved in catalysis have been identified on the basis of the three-dimensional structure. Site-directed mutants have been used to further characterize the role of these residues in binding and catalysis. A chemical mechanism for the enzyme, based on these and previously reported data, is proposed.     

Structure-based design of a potent transition state analogue for TEM-1 beta-lactamase

The structure of the plasmid-mediated beta-lactamase TEM-1 has been solved in complex with a designed boronic acid inhibitor (1R)-1-acetamido-2-(3-carboxyphenyl)ethane boronic acid at 1.7 A resolution. The boronate inhibitor was designed based on the crystallographic coordinates of the acyl-enzyme intermediate of TEM-1 bound to the substrate penicillin G. The boronate-TEM-1 complex is highly ordered and defines a novel transition state analogue of the deacylation step in the beta-lactamase reaction pathway. The design principles of this highly effective inhibitor (Ki = 110 nM) and the resulting structural and mechanistic implications are presented.     

Crystal structure of an engineered Cro monomer bound nonspecifically to DNA: possible implications for nonspecific binding by the wild-type protein

The structure has been determined at 3.0 A resolution of a complex of engineered monomeric Cro repressor with a seven-base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half-operator that is recognized by each subunit of the wild-type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence-independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48 degrees further out of the major groove, while the turn region of the helix-turn-helix remains in contact with the DNA backbone. All of the direct base-specific interactions seen in the wild-type Cro-operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein DNA interface than for half of the wild-type Cro-operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence-nonspecific interaction of the wild-type Cro dimer with DNA. In the sequence-specific complex, the DNA is bent, the protein dimer undergoes a large hinge-bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.     

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.     

Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli

UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) is a cytoplasmic enzyme involved in the biosynthesis of peptidoglycan which catalyzes the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA). The crystal structure of MurD in the presence of its substrate UMA has been solved to 1.9 A resolution. Phase information was obtained from multiple anomalous dispersion using the K-shell edge of selenium in combination with multiple isomorphous replacement. The structure comprises three domains of topology each reminiscent of nucleotide-binding folds: the N- and C-terminal domains are consistent with the dinucleotide-binding fold called the Rossmann fold, and the central domain with the mononucleotide-binding fold also observed in the GTPase family. The structure reveals the binding site of the substrate UMA, and comparison with known NTP complexes allows the identification of residues interacting with ATP. The study describes the first structure of the UDP-N-acetylmuramoyl-peptide ligase family.     

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.     

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli

Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C beta-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C beta-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C beta-lactamases, AmpH showed no beta-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the beta-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.     

Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents

The three-dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8-residue insertion near the first disulfide (Cys 45-Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7-residue deletion replacing helix hN2 (Ser 110-Tyr 114), which enlarges the S3 pocket; (3) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and (4) an ordered 11-residue addition at the carboxy terminus. The inhibitor binds in an extended conformation and presents a branched structure at the P3 position. The implications of these findings for the design of potent antifungal agents are discussed.     

Transcontinental importation into the UK of Escherichia coli expressing a plasmid-mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended-spectrum beta-lactamase in a Leeds hospital

Sixteen strains of Escherichia coli with high-level resistance to extended-spectrum cephalosporins and other classes of antibiotic have been isolated at St James' University Hospital, Leeds. They produce up to three separate beta-lactamases: TEM-1, SHV-5 and, in five isolates, a plasmid-mediated AmpC-type enzyme. With the exception of carbapenems, the isolates reported in this study were resistant to all beta-lactam antibiotics including extended-spectrum cephalosporins and the monobactam aztreonam. There was evidence of the spread of a plasmid encoding SHV-5, particularly amongst patients on the liver transplant unit. Sensitivity to beta-lactam antibiotics in five isolates expressing the AmpC-type beta-lactamase was not restored by the beta-lactamase inhibitor clavulanic acid. These bacteria also carried blaSHV-5 on a large plasmid. PCR-amplification of the structural gene and digestion with restriction endonucleases demonstrated that the plasmid-mediated blaAmpC probably identified as BIL-1 using the criteria available. Four of the five patients carrying isolates that carried the plasmid-located blaAmpC gene had recently visited the Indian subcontinent and we presume that they returned carrying these bacteria. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis (PFGE) suggests that at least four distinct strains existed amongst these five isolates. The two isolates that had very similar PFGE patterns had different plasmid profiles and were isolated from different locations in the hospital and at different times. This study demonstrates the ease with which highly resistant bacteria can be imported into the UK and spread within hospitals.     

Three-dimensional reconstruction with contrast transfer function correction from energy-filtered cryoelectron micrographs: procedure and application to the 70S Escherichia coli ribosome

Cryoelectron microscopy provides the means of studying macromolecules in their native state. However, the contrast transfer function (CTF) makes the images and the three-dimensional (3D) maps derived from them difficult to interpret. We developed methods to determine the CTF from experimental data and to obtain a CTF-corrected 3D reconstruction. The CTF correction and 3D reconstruction accomplished in one step make it easy to combine different defocus data sets and decrease the error accumulation in the computation. These methods were applied to energy-filtered images of the 70S Escherichia coli ribosome, resulting in a distortion-free 3D map of the ribosome at 1/24.5 A-1 resolution, as determined by the differential phase residual resolution criterion.     

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.     

Primary structure and possible functions of a trypsin inhibitor of Bombyx mori

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two-dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 x 10-10), but does not alpha-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.     

Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.     

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.     

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.     

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.     

Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.