Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min−1 and a KI of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with β-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The β-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its β-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the β-elimination product of oCTP.

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., α-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 → Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.

A resonant mirror biosensor was used to study cyclic nucleotide–receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Iα regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (K′i = KicAMP/Kianalog) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published K′i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K′i values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide–receptor interactions.

The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq1 = 6.85 × 109 M−1 s−1 and kq2 = 1.88 × 109 M−1 s−1, that merge into one in the complex with kq = 1.78 × 109 M−1 s−1.

500 ps trajectory analysis of Trp χ1/χ2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between χ1/χ2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

Little is known of the plant branched-chain α-ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size-exclusion column to >90% homogeneity and was catalytically active. The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24-mer. Negative-staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3–84 were 0.76 ± 0.14 Å for the backbone atoms and 1.36 ± 0.15 Å for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 Å, respectively, for residues 4–83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H–1H–13C NOESY-HSQC spectra with and without 13C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.

The crystal structure of the fibrinolytic enzyme tissue plasminogen activator (tPA) shows that the bulky side chain of Y99 hinders access to the active site by partially occluding the S2 site and may be responsible for the low catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient proteases like trypsin and thrombin. The Y99L replacement results in an increase in the kcat/Km for chromogenic substrates due to enhanced diffusion into the active site. The increase is modest (threefold) for substrates specific for tPA that carry Pro or Gly at P2, but reaches 80-fold for less specific substrates carrying Arg at P2. On the other hand, the Y99L mutation has no effect on the activity of tPA toward the natural substrate plasminogen, that carries Gly at P2, and reduces more than 10-fold the inhibition of tPA by plasminogen activator inhibitor-1 (PAI-1), that carries Ala at P2. We conclude that the steric hindrance provided by Y99 in the crystal structure affects mostly nonphysiological substrates with bulky residues at P2. In addition, residue Y99 plays an active role in the recognition of PAI-1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibition by PAI-1 without compromising the fibrinolytic potency of tPA, a result of potential therapeutic relevance.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent—2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels α-lactalbumin as a function of pH (3–7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23–34) and C (86–98) helix of the native structure are extensively labeled. The small β-domain (40–50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of α-lactalbumin, the α-domain has a greater degree of persistent structure than the β-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

Previous investigation has shown that at 22 °C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512–525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, ∼80–90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0–50 μM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 °C reaches a limiting value of 0.014 s−1. For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s−1, suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.

In a recent press release, the British MRC announced a historic day in scientific history, the completion of the Ceanorhebtitis elegans genome. This first genetic blueprint of an animal adds more than 19,000 genes to the several hundred thousand already available. The sequence databases are regarded as a treasure store. Genome data mining shows signs of triggering a gold rush.

Transmembrane helices are the most readily predictable secondary structure components of proteins. They can be predicted to a high degree of accuracy in a variety of ways. Many of these methods compare new sequence data with the sequence characteristics of known transmembrane domains. However, the known transmembrane sequences are not necessarily representative of a particular organism. We attempt to demonstrate that parameters optimized for the known transmembrane domains are far from optimal when predicting transmembrane regions in a given genome. In particular, we have tested the effect of nucleotide bias upon the composition and hence the prediction characteristics of transmembrane helices. Our analysis shows that nucleotide bias of a genome has a strong and predictable influence upon the occurrences of several of the most important hydrophobic amino acids found within transmembrane helices. Thus, we show that nucleotide bias should be taken into account when determining putative transmembrane domains from sequence data.

Methods for protein structure (3D)–sequence (1D) compatibility evaluation (threading) have been developed during the past decade. The protocol in which a sequence can recognize its compatible structure in the structural library (i.e., the fold recognition or the forward-folding search) is available for the structure prediction of new proteins. However, the reverse protocol, in which a structure recognizes its homologous sequences among a sequence database, named the inverse-folding search, is a more difficult application. In this study, we have investigated the feasibility of the latter approach. A structural library, composed of about 400 well-resolved structures with mutually dissimilar sequences, was prepared, and 163 of them had remote homologs in the library. We examined whether they could correctly seek their homologs by both forward- and inverse-folding searches. The results showed that the inverse-folding protocol is more effective than the forward-folding protocol, once the reference states of the compatibility functions are appropriately adjusted. This adjustment only slightly affects the ability of the forward-folding search. We noticed that the scoring, in which a given sequence is re-mounted onto a structure according to the 3D–1D alignment determined by the dynamic programming method, is only effective in the forward-folding protocol and not in the inverse-folding protocol. Namely, the inverse-folding search works significantly better with the score given by the 3D–1D alignment per se, rather than that obtained by the re-mounting. The implications of these results are discussed.

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2′-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2′-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2′-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2′-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of ∼100 (p21ras) or ∼105 (Rab5), and the resulting equilibrium dissociation constants are in the nm or μM range, respectively. The data demonstrate that 2′halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 105-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the μM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one-fourth as well. It was shown that the N-terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N-terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co-translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of an Arg-Gly-Pro helix linker segment (residues 11–14) from the L2V mutant, which shifted three leucine residues toward the N-terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N-termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.

We have used differential scanning calorimetry to determine the effect of low concentrations (C = 0–2 M) of the osmolyte sarcosine on the Gibbs energy changes (ΔG) for the unfolding of hen-egg-white lysozyme, ribonuclease A, and ubiquitin, under the same buffer and pH conditions. We have also computed this effect on the basis of the additivity assumption and using published values of the transfer Gibbs energies for the amino acid side chains and the peptide backbone unit. The values thus predicted for the slope ∂ΔG/∂C agree with the experimental ones, but only if the unfolded state is assumed to be compact (that is, if the accessibility to solvent of the unfolded state is modeled using segments excised from native structures). The additivity-based calculations predict similar ∂ΔG/∂C values for the three proteins studied. We point out that, to the extent that this approximate constancy of ∂ΔG/∂C holds, osmolyte-induced increases in denaturation temperature will be larger for proteins with low unfolding enthalpy (small proteins that bury a large proportion of apolar surface). The experimental results reported here are consistent with this hypothesis.

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1H NMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Hα secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N-Cα-C=O) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06 ± 0.30 Å using residues V4–P35 and 0.51 ± 0.17 Å using residues A7–S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and “snorkeling” of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

δ-Crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two δ crystallins, denoted δ1 and δ2. Duck δ2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to δ2/ASL in the amino acid sequence, δ1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras—221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, δ1 or δ2, respectively—were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to δ1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in δ1. The thermodynamic characterization of human ASL (hASL) and duck δ1 and δ2 indicate that δ crystallins are slightly less stable than hASL, with the δ1 being the least stable. The ΔGs of unfolding are 57.25, 63.13, and 70.71 kcal mol−1 for δ1, δ2, and hASL, respectively. This result was unexpected, and we speculate that δ crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein–protein and protein–solvent interactions.

The importance of unsatisfied hydrogen bonding potential on protein–protein interaction was studied. Two alternate modes of dimerization (conventional and flipped form) of an immunoglobulin light chain variable domain (VL) were previously identified. In the flipped form, interface residue Gln89 would have an unsatisfied hydrogen bonding potential. Removal of this Gln should render the flipped dimer as the more favorable quaternary form. High resolution crystallographic studies of the Q89A and Q89L mutants show, as we predicted, that these proteins indeed form flipped dimers with very similar interfaces. A small cavity is present in the Q89A mutant that is reflected in the ∼100 times lower association constant than found for the Q89L mutant. The association constant of Q89A and Q89L proteins (4 × 106 M−1 and >108 M−1) are 10- and 1,000-fold higher than that of the wild-type protein that forms conventional dimers clearly showing the energetic reasons for the flipped dimer formation.

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53–Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly α-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53–Gly69 of cAcP indicate that only the native-like trans isomer of the Gly–Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an ∼1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53–Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5′-triphosphate (PTP), 6-chloropurine 5′-triphosphate (Cl-PTP), 6-mercaptopurine 5′-triphosphate (SH-PTP), 6-methylpurine 5′-triphosphate (Me-PTP), and 1-methyladenosine 5′-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of l-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2′ and 3′ ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2′-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.