Binding Kinetic Constants Research Articles

Kinetic, Affinity, and Diversity Limits of Human Polyclonal Antibody Responses against Tetanus Toxoid

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 x 10(-3) s(-1). Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.

Metal‐chelating affinity hydrogels for sustained protein release

Affinity hydrogels based on poly(ethylene glycol) diacrylate and a metal-ion-chelating ligand, glycidyl methacrylate-iminodiacetic acid, have been developed to systematically decrease protein release rates from hydrophilic tissue engineering scaffolds formed in situ. In the current work, tunable and sustained release of a model protein, hexa-histidine tagged green fluorescence protein (hisGFP), is accomplished by judiciously increasing ligand:protein ratio or replacing low-affinity nickel ions with high-affinity copper ions. Agreement between theoretical predictions of a reaction-diffusion model and experimental measurements confirm metal- ion-mediated sustained protein release from these affinity hydrogels is governed by equilibrium protein-ligand binding affinity (dissociation constant, Kd) as well as protein-ligand dissociation kinetics (protein debinding rate constant, k off). The former dictates the release rate in the early period of protein release while the latter determines the long-term sustained release effect. While metal-ion affinity binding has been widely used for various purposes including protein purification and surface patterning, this is the first report describing its application in systematically controlling protein release from hydrophilic PEG networks suitable for cell encapsulation. By using ligands with proper binding kinetic constants (Kd and k off), localized protein delivery can be sustained over clinically relevant timescales while maintaining a favorable environment for cell encapsulation and viability.

Porous SiO2Interferometric Biosensor for Quantitative Determination of Protein Interactions: Binding of Protein A to Immunoglobulins Derived from Different Species

Determination of kinetic and thermodynamic protein binding constants using interferometry from a porous Si Fabry-Perot layer is presented. A protein A capture probe is adsorbed within the pores of an oxidized porous Si matrix, and binding of immunoglobulin G (IgG) antibodies derived from different species is investigated. The relative protein A/IgG binding affinity is human > rabbit > goat, in agreement with literature values. The equilibrium binding constant (Ka) for human IgG binding to surface-immobilized protein A is determined to be (3.0 +/- 0.5) x 107 M-1 using an equilibrium Langmuir model. Kinetic rate constants are calculated to be kd = (2.1 +/- 0.2) x 10-4 s-1 and ka = (1.2 +/- 0.4) x 104 M-1 s-1 using nonlinear least-squares analysis, yielding an equilibrium binding constant of Ka = (5.5 +/- 1.5) x 107 M-1. Both steady-state and time-dependent measurements yield equilibrium binding constants that are consistent with literature values. Kinetic rate constants determined through nonlinear least-squares analysis are also in agreement with protein A/IgG binding on a surface. Dosing with a high concentration of analyte leads to deviations from ideal binding behavior, interpreted in terms of restricted analyte diffusion within the porous SiO2 matrix. It is shown that the diffusion limitations can be minimized if the kinetic measurements are performed at low analyte concentrations or under conditions in which the protein A capture probe is not saturated with analyte. Potential limitations of the use of porous SiO2 interferometers for quantitative determination of protein binding constants are discussed.

Restraining the conformation of HIV-1 gp120 by removing a flexible loop

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.

Role of Phenylalanine B10 in Plant Nonsymbiotic Hemoglobins,

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.

Characterization of Binding of Cholinesterases to Surface Immobilized Ligands

We summarize here the development of various piezoelectric biosensors utilizing cholinesterase (ChE) as the recognition element. In our work we studied the interaction between cholinesterase and its ligands (propidium, carnitine, benzylgonine‐1,8‐diamino‐3,4‐dioxaoctane (BZE‐DADOO) and paraoxon). The sensor modification was based on a self‐assembled monolayer (SAM) of a thiol compound (11‐mercaptoundecanoic acid) on the gold electrode and the subsequent covalent coupling of the cholinesterase ligand to this SAM. The ligand‐modified piezoelectric sensors were placed in a flow system to allow the on‐line monitoring of cholinesterase binding and the enzymatic activity quantification by amperometry. Cholinesterases from different species—acetylcholinesterase (AChE) from Electrophorus electricus, AChE from Drosophila melanogaster, and butyrylcholinesterase (BChE) of human origin—were tested on the various immobilized ligands. Our research allowed the development of a competitive assay for the detection of organophosphates in river water samples using the BZE‐DADOO‐modified piezosensor. Another direction of research was pointed on the characterization of the interactions between ChE and its ligands. The kinetic binding constants were derived using a one‐to‐one binding model.

The Structure of the Interleukin-15α Receptor and Its Implications for Ligand Binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.

Real time kinetic analysis of the interaction between interleukin-1α and soluble interleukin-1 receptor I using a resonant mirror biosensor

A novel method for real time kinetic analysis of the interaction between IL-1α and sIL-1R I is reported. sIL-1R I was immobilized on the biotin cuvette surface of the resonant mirror biosensor to set up the experimental model. Results obtained from the assay confirmed that biotinylated sIL-1R I was specifically immobilized on the avidin-coated biotin cuvette surface, the interaction between IL-1α and sIL-1R I was fast and specific, and the interaction response is dose-dependence of IL-1α in solution with a range of 150–2400 nM. The binding curve was fitted by FASTfit analysis, which fitted the monophasic association quite well, and the error did not exceed 0.4 arc seconds. Kinetic constants for IL-1α binding to sIL-1R I were determined from the plots of K on versus the concentration of IL-1α. For the interaction, k ass was 2.81 × 10 3 M −1 s −1, K diss was 2.52 × 10 −3 s −1, and K D was 8.97 × 10 −7 M.

Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

The H Box-harboring Domain Is Key to the Function of the Salmonella enterica PhoQ Mg2+-sensor in the Recognition of Its Partner PhoP

In two-component signaling systems, the transduction strategy relies on a conserved His-Asp phosphoryl exchange between the sensor histidine kinase and its cognate response-regulator, and structural and functional consensus motifs are found when comparing either the diverse histidine kinases or response regulators present in a single cell. Therefore, the mechanism that guarantees the specific recognition between partners of an individual pair is essential to unequivocally generate the appropriate adaptive response. Based on sequence alignments with other histidine kinases, we dissected the Salmonella enterica Mg2+-sensor PhoQ in different subdomains and examined by in vivo and in vitro assays its interaction with the associated response regulator PhoP. This signal transduction system allows Salmonella to withstand environmental Mg2+ limitation by triggering gene expression that is vital throughout the infective cycle in the host. Using resonant mirror biosensor technology, we calculated the kinetic and equilibrium binding constants and determined that the His-phosphotransfer domain is essential for the PhoQ specific recognition and interaction with PhoP. Additionally, we show the role of this domain in the bimolecular transphosphorylation and provide evidence that this region undergoes dimerization.

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding.

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.

Frontal Affinity Chromatography Coupled to Mass Spectrometry for Screening Mixtures of Enzyme Inhibitors

Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 μL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Glcp-OR acceptor giving β-d-GlcpNAc-(1→2)-[β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)-β-d-Glcp-OR as the reaction product. The kinetic parameters Km and Vmax for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded Kd values for each of the eight compounds ranging from 0.3 to 36 μM. These Kd values were 2 to 10 times lower than the inhibition constants, KI's, determined in solution using a standard radiochemical assay. However, the ranking order of Kd's was the same as the ranking of KI values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor Kd values making it a valuable tool for enzyme inhibitor evaluations.

A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors.

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.

A Homogeneous Method to Measure Aminoacyl-tRNA Synthetase Aminoacylation Activity Using Scintillation Proximity Assay Technology

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.

Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor by reducing the microscopic agonist dissociation constant.

Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine. The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization. Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.

Differential antibody reactivity and CD4 binding of the mammary tumor marker protein GCDFP-15 from breast cyst and its counterparts from exocrine epithelia.

Analysis of biopsies from breast cancer patients demonstrated that GCDFP-15 (gross cystic disease fluid protein-15) is a specific immunocytochemical marker of primary and secondary apocrine breast tumors. The protein has an amino acid sequence identical to SABP (secretory actin-binding protein), to PIP (prolactin-inducible protein) and to gp17, a protein isolated from human seminal plasma. The latter was found to bind to CD4, a T-cell co-receptor involved in antigen recognition, thereby inhibiting the ability of the receptor to interact with the HIV-1 envelope protein gp120. We compare here the ability of independently purified GCDFP-15, SABP and gp17 and of recombinant PIP both to cross-react with a panel of monoclonal antibodies (MAbs) raised against GCDFP-15 or gp17, respectively, and to bind to CD4. We show that, although the various factors share the ability to bind to the panel of antibodies used, differences in the pattern of MAb recognition can be demonstrated. By comparing the kinetic constants for binding of GCDFP-5 and gp17 to CD4 by biosensor technology, significant differences in binding affinities were observed between the 2 factors, thus reflecting structural differences. Surface plasmon resonance analysis also showed that anti-GCDFP-15 and anti-gp17 antibodies inhibit the binding of CD4 to GCDFP-15 and gp17, respectively, to different extents. Our data thus indicate that, while the various forms of the protein are encoded by the same cDNA, tissue specificities due to post-translational modifications exist. This information may be relevant for developing more sensitive and accurate tests for the use of GCDFP-15 as a diagnostic mammary tumor marker and, most importantly, raises the possibility that GCDFP-15 may constitute a breast tumor-specific antigen.

Magnesium and calcium chelation by a bis-spiropyran

A bis-benzospiropyranindoline was prepared by a simple two-step procedure. The magnesium and calcium chelating ability of this photochromic spiropyran was investigated and compared to simple mono-spiropyrans. Kinetic binding constants were measured. Moderately strong metal binding occurs in acetone solution ( K=40 000 M −1 for Mg, K=13 000 M −1 for Ca) when the bis-spiropyran is irradiated with light at 365 nm. This binding is eight times higher than the binding of the mono-spiropyrans studied. The color of the merocyanine form of the bis-spiropyran ( λ max=548 nm) is strongly influenced by the metal, blue-shifting the maximum absorbance 43 nm (Mg) and 22 nm (Ca). Strong fluorescence is observed when the bis-spiropyran complexed to either metal is irradiated at 365 nm, with emission maxima of 586 nm (Mg) and 606 nm (Ca). The strength of the binding is inversely correlated to the unimolecular decomposition rate constant of the spiropyran–metal complex. The fluorescence emission maxima become increasingly blue-shifted as the strength of the binding increases. The fluorescence is compared to the metal-free spiropyran, as well as to simple mono-spiropyrans coordinated to calcium. The mechanism of decoloration of the bis-spiropyran with and without metals present is discussed.

Functional properties of the molecular chaperone DnaK from Thermus thermophilus

The genes coding for the Thermus thermophilus (Tth) homologues of the molecular chaperones DnaK and GrpE (DnaK Tthand GrpE Tth) were cloned and expressed in Escherichia coli. The proteins were purified and their functional properties were assessed by equilibrium and transient kinetic methods. DnaK Tthhas an intrinsic ATPase activity of 3×10 −4s −1at 25°C and 10×10 −4 s −1at 75°C under single turnover conditions. It binds the fluorescent nucleotide analogue N 8-(4- N′-methylanthraniloylaminobutyl)-8-aminoadenosine 5′-diphosphate (MABA-ADP) with a dissociation constant ( K d) of 3 nM and ADP with a K dof 47 nM at 25°C. At 75°C the affinities are decreased fivefold to 15 nM (MABA-ADP) and 280 nM (ADP). The kinetic constants for two-step binding of MABA-ADP and of ADP to DnaK Tthwere determined at 25°C and 75°C, respectively. GrpE Tthacts as a nucleotide-exchange factor on DnaK Tthand accelerates the release of bound MABA-ADP significantly. This shows that the nucleotide-binding domain is functionally intact, and that the specific interaction of DnaK Tthand GrpE Tthis mediating nucleotide exchange. A fluorescently labelled peptide that comprises a subsequence of the E. coli transcription factor σ 32binds to nucleotide-free DnaK Tthwith a K dof 4.9 μM. Displacement with unlabelled peptide yields a K dof 5.0 μM for the unlabelled peptide. Thus the peptide-binding domain also appears to be functional. For the cellular chaperone function of DnaK, a coupling between nucleotide and peptide-binding domains is required. However, with DnaK Tthin the ATP as well as in the ADP.P i-state, peptide is bound and released within seconds. No correlation between ATP-binding or hydrolysis by DnaK Tthand changes in the σ 32peptide exchange rates could be detected. It thus appears that the DnaK system from Th. thermophilus has a different mechanism of coupling the nucleotide state to the fast and slow peptide exchange properties.

Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.

Conformational Integrity and Ligand Binding Properties of a Single Chain T-cell Receptor Expressed in Escherichia coli

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.