Analysis of phi and chi 1 torsion angles for hen lysozyme in solution from 1H NMR spin-spin coupling constants

Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J alpha beta coupling constants have been measured for 57 residues of the 129-residue protein hen egg white lysozyme. These NMR data have been compared with torsion angles defined in the tetragonal and the triclinic crystal forms of the protein. For most residues the measured 3JHN alpha values were consistent with the phi torsion angles found in both crystal forms; the RMS difference between the coupling constants calculated by using the tetragonal crystal structure phi angles and the experimental 3JHN alpha values is 0.88 Hz. Thus there appears to be no significant averaging of the phi torsion angle either in the interior or at the surface of the protein. For 41 of the residues where 3J alpha beta coupling constants have been determined, the values are consistent with a single staggered conformation about the chi 1 torsion angle and there is complete agreement between the NMR data in solution and the torsion angles defined in the crystalline state. In contrast, for the other 16 residues where 3J alpha beta coupling constant values have been measured, the data indicate extensive motional averaging about the chi 1 torsion angle. These residues occur largely on the surface of the protein and examination of the crystal structures shows that many of these residues adopt a different conformation in the triclinic and tetragonal crystal forms and have high crystallographic temperature factors. It appears, however, that in solution conformational flexibility of the side chains of surface residues is significantly more pronounced than in individual crystal structures.     

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

A study of the lysyl residues in the basic pancreatic trypsin inhibitor using 1H nuclear magnetic resonance at 360 Mhz.

Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Nepsilon-dimethyllsysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine-41 is involved in an intramolecular interaction with tyrosine-10. Since in the single crystal structure tyrosine-10 is involved in an intermolecular interaction with arginine-42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.     

Methotrexate binds in a non-productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy

Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexate. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.     

Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.     

Environmentally induced conformational changes in B-type DNA: comparison of the conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its crystalline complex with the restriction nuclease EcoRI

Raman spectroscopic analysis of the secondary structure of the crystalline restriction endonuclease EcoRI, the oligonucleotide d(TCGCGAATTCGCG) in solution, and the corresponding crystalline EcoRI-oligonucleotide complex reveals structural differences between the complexed and uncomplexed protein and oligonucleotide components that appear to be linked to complex formation. Structural differences that are spectroscopically identified include (1) an increase in the population of furanose rings adopting the C3'-endo conformation and (2) spectroscopically observed changes in base stacking which are probably associated with the crystallographically observed distortion of the phosphate backbone about positions C(3)-G(4) and C(9)-G(10) and unwinding between the symmetry-related segments GAA-TTC which make up the central recognition core (McClarin et al., 1986). Changes in base stacking due to distortions and unwinding along the oligonucleotide result in differences in the base vibrational region between the spectra of the complex and the oligonucleotide in solution. The spectroscopic analysis indicates that the C2'-endo population is similar for the oligonucleotide in solution and in the complex. The additional C3'-endo population in the complex appears to arise from the conversion of rings adopting alternative conformations such as C1'-exo and O1'-endo. Analysis of the vibrational bands derived from guanine indicates that the population of guanine residues associated with furanose rings in a C2'-endo conformation is similar for the oligonucleotide in solution and in the crystalline complex. This implies that the increase in C3'-endo population is not associated with guanine residues. Large conformational distortions such as those observed in the crystal distortions are not observed in either the crystal or the solution of the oligomer d(CGCGAATTCGCG).(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix alpha3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the "coupling of binding and folding" phenomenon.     

Conformation of endothelin in aqueous ethylene glycol determined by 1H-NMR and molecular dynamics simulations

The solution conformation of a 21-residue vasoconstrictor peptide endothelin-1 (ET-1) in water-ethylene glycol has been determined by two-dimensional 1H-NMR spectroscopy and constrained molecular dynamics simulations. The N-terminus (residues 1-4) appears to undergo conformational averaging and no single structure consistent with the NMR constraints could be found for this region. Residues 5-8 form a turn, and residues 9-16 exist in a helical conformation. A flexible 'hinge' between residues 8-9 allows various orientations of the turn relative to the helix. Another 'hinge' at residue 17 connects the extended C-terminus to the bicyclic core region (residues 1-15). Residues important for binding and biological activity form a contiguous surface on one side of the helix, with the two disulfides extending from the other side of the helix.     

Charge-transfer studies of the availability of aromatic side chains of proteins in guanidine hydrochloride.

The ability of aromatic tryptophyl and tyrosyl side-chain donors to form charge-transfer (CT) complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride has been used to investigate the degree of exposure of these aromatic residues in denaturated proteins. The coplanar geometry of the CT complexes requires that virtually a full ring face of the donor be available for interaction with the acceptor, and the aromatic donor residues of lysozyme, trypsin, chymotrypsin, and the zymogens of the latter two enzymes do not appear to be wholly "exposed" in 6 M guanidine hydrochloride. Comparison of the CT proerties of the proteins with the corresponding properties of model complexes suggests that the incomplete exposure is due at least in part to statistical fluctuations in the continuously mobile, randomly coiled polypeptide chain which result in residues being alternately fully exposed and partly covered. Reduction and alkylation of the disulfide cross-links increase the apparent availability of the aromatic residues but the exposure is still less than that expected from a comparable mixture of tryptophan and tyrosine residues. Previous studies on the exposure of the aromatic residues of lysozyme and trypsin in aqueous salt solutions, when taken together with the present results, further suggest that there are two distinct kinds of surface environment possible on native proteins in solution. Some residues appear to be located in areas of the protein surface which are characterized by relatively fixed or stable local conformations, and have apparent CT association constants closely resembling these of comparable model complexes. Other residues may be located in a region where the protein conformation is flexible or continuously mobile, as evidenced by their smaller apparent association constants. It is probably significant that Trp-62 of lysozyme and Trp-215 of trypsin, both specificity site residues, appear to belong to the class of residues which can be considered as being in a flexible environment on the protein surface.     

Streptokinase is a flexible multi-domain protein

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.     

Flexibility analysis of enzyme active sites by crystallographic temperature factors

Protein flexibility is inherent to protein structural behavior. Experimental evidence for protein flexibility is extensive both in solution and in the solid state. A major question is whether the flexibility observed in enzymes is simply an inherent property of proteins that must always be borne in mind or is essential for catalysis or substrate binding. The temperature factors or B-values, as determined crystallographically, are linearly related to the mean square displacement of an atom and give an indication of atomic flexibility in the crystalline state. In this paper, we describe the frequency distributions of the normalized B-factor (B'-factor) for the active site and non-active site residues in the selected 69 apo-enzymes. This analysis was performed over the entire sequences and for different structural subsets defined by the three-dimensional structure of proteins, as alpha-helices, beta-structures and coil conformation and buried and non-buried residues. The results show that in all cases, the active site residues predominantly occur in region of low B'-factor and the non-active site residues have a tendency to exist in the high B'-factor region. This observation suggests that the active site residues, in general, are less flexible than the non-active site residues and therefore the vibrational and the fast collective motions of the C(alpha) atoms of proteins appear not to have clear biological significance.     

Structure of crambin in solution, crystal and in the trajectories of molecular dynamics simulations

The mechanisms of the three-dimensional crambin structure alterations in the crystalline environments and in the trajectories of the molecular dynamics simulations in the vacuum and crystal surroundings have been analyzed. In the crystalline state and in the solution the partial regrouping of remote intramolecular packing contacts, involved in the formation and stabilization of the tertiary structure of the crambin molecule, occurs in NMR structures. In the crystalline state it is initiated by the formation of the intermolecular contacts, the conformational influence of its appearance is distributed over the structure. The changes of the conformations and positions of the residues of the loop segments, where the intermolecular contacts of the crystal surroundings are preferably concentrated, are most observable. Under the influence of these contacts the principal change of the regular secondary structure of crambin is taking place: extension of the two-strand β structure to the three-strand structure with the participation of the single last residue N46 of the C-terminal loop. In comparison with the C-terminal loop the more profound changes are observed in the conformation and the atomic positions of the backbone atoms and in the solvent accessibility of the residues of the interhelical loop. In the solution of the ensemble of the 8 NMR structures relative accessibility to the solvent differs more noticeably also in the region of the loop segments and rather markedly in the interhelical loop. In the crambin cryogenic crystal structures the positions of the atoms of the backbone and/or side chain of 14–18 of 46 residues are discretely disordered. The disorganizations of at least 8 of 14 residues occur directly in the regions of the intermolecular contacts and another 5 residues are disordered indirectly through the intramolecular contacts with the residues of the intermolecular contacts. Upon the molecular dynamics simulation in the vacuum surrounding as in the solution of the crystalline structure of crambin the essential changes of the backbone conformation are caused by the intermolecular contacts absence, but partly masked by the structure changes owing to the nonpolar H atoms absence on the simulated structure. The intermolecular contact absence is partly manifested upon the molecular dynamics simulation of the crambin crystal with one protein molecule. Compared to the crystal structure the lengths of the interpeptide hydrogen bonds and other interresidue contacts in an average solution NMR structure are somewhat shorter and accordingly the energy of the interpeptide hydrogen bonds is better. This length shortening can occur at the stage of the refinement of the NMR structures of the crambin and other proteins by its energy minimizations in the vacuum surroundings and not exist in the solution protein structures.     

Protein conformer selection by sequence-dependent packing contacts in crystals of 3-phosphoglycerate kinase

In several crystal structures of 3-phosphoglycerate kinase (PGK), the two domains occupy different relative positions. It is intriguing that the two extreme (open and closed) conformations have never been observed for the enzyme from the same species. Furthermore, in certain cases, these different crystalline conformations represent the enzyme-ligand complex of the same composition, such as the ternary complex containing either the substrate 3-phosphoglycerate (3-PG) and beta,gamma-imido-adenosine-5'-triphosphate (AMP-PNP), an analogue of the substrate MgATP, or 3-PG and the product MgADP. Thus, the protein conformation in the crystal is apparently determined by the origin of the isolated enzyme: PGK from pig muscle has only been crystallized in open conformation, whereas PGK from either Thermotoga maritima or Trypanosoma brucei has only been reported in closed conformations. A systematic analysis of the underlying sequence differences at the crucial hinge regions of the molecule and in the protein-protein contact surfaces in the crystal, in two independent pairs of open and closed states, have revealed that 1) sequential differences around the molecular hinges do not explain the appearance of fundamentally different conformations and 2) the species-specific intermolecular contacts between the nonconserved residues are responsible for stabilizing one conformation over the other in the crystalline state. A direct relationship between the steric position of the contacts in the three-dimensional structure and the conformational state of the protein has been demonstrated.     

Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: a high resolution structural characterization of a peptide model in aqueous solution.

There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.     

Tritium planigraphy: Differences in the spatial structures of the influenza virus M1 protein in crystal,solution, and virion

The structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) in solution at acidic pH and in the composition of the virion has been studied by the tritium planigraphy method. A model of the spatial structure was constructed using a special algorithm simulating the experiment and a set of algorithms for predicting the secondary structure and disordered regions in proteins. The tertiary structure was refined using the Rosetta program. For a comparison of the structures in solution and inside the virion, the data of X-ray diffraction analysis for the NM domain were also used. The main difference in the structures of the protein in solution and the crystalline state is observed in the region of contact of N and M domains, which in the crystalline state is packed more densely. The regions of the maximum label incorporation almost completely coincide with unstructured regions in the protein that were predicted by the bioinformatics analysis. These regions are concentrated in the C domain and in loop regions between M, N, and C domains. The data were confirmed by analytical centrifugation and dynamic light scattering. Anomalous hydrodynamic dimensions and a low structuration of the M1 protein in solution were found. The polyfunctionality of the protein in the cell is probably related to its flexible tertiary structure, which, owing to unstructured regions, provides contact with various partner molecules.     

Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility,conformation and mapping of contact regions

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.     

1H NMR studies of bovine and porcine phospholipase A2: assignment of aromatic resonances and evidence for a conformational equilibrium in solution

Bovine and porcine pancreatic phospholipases A2, and porcine isophospholipase A2, have been investigated by one- and two-dimensional 1H NMR spectroscopy. Resonances have been assigned for 20-26 residues in each enzyme, including all the aromatic residues, by a strategy based on the semiquantitative comparison of proximity relationships deduced from NOE experiments with those seen in the crystal structure NOE experiments indicate that the loop comprising residues 59-70, which has a different conformation in the crystal structures of the bovine and porcine enzymes, has the same conformation in these two enzymes in solution. Selective changes in the line width of a limited number of resonances as a function of pH, temperature, and calcium concentration provide evidence for a local conformational equilibrium. This equilibrium involves a limited region of the protein structure around residues 25, 41, 106, and 111; it has been identified in the bovine enzyme and porcine isoenzyme but is not apparent in the porcine enzyme.     

Analysis of secondary structural and physicochemical changes in protein–protein complexes

Conformation switching in protein–protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein–protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein–protein modeling and docking studies.     

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.     

NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors.

The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.