Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. I. Evidence for ion-induced conformational change

Further insight into the cosubstrate-induced structural change of the melibiose permease (MelB) of Escherichia coli has been sought by investigating the binding and spectroscopic properties of the fluorescent sugar 2'-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl 1-thio-beta-D-galactopyranoside (Dns2-S-Gal) and related analogs (Dns3-S-Gal or Dns6-S-Gal with a propyl or hexyl instead of an ethyl linker, respectively) interacting with MelB in membrane vesicles or in proteoliposomes. The three analogs efficiently inhibit melibiose transport and bind to MelB in a sodium-dependent fashion. Their dissociation constants (Kd) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the presence of NaCl and Dns2-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns3-S-Gal or Dns6-S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detailed study of the Dns2-S-Gal signal elicited by a 297 nm illumination indicates that a tryptophan-mediated fluorescence resonance energy transfer phenomenon is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants describing their dependence on the probe or NaCl concentrations closely correlate with the probe binding constants. Finally, the Dns2-S-Gal signal recorded in sodium-free medium is red shifted by up to 25 nm from that recorded in the presence of NaCl. Taken together, these results suggest (i) that the fluorescence signals below 500 nm arise from Dns-S-Gal molecules bound to MelB, (ii) the presence of a highly hydrophobic environment close to or at the sugar-binding site, the polarity of which increases on moving away from the sugar-binding site, and (iii) that the interaction of sodium ions with MelB enhances the hydrophobicity of this environment. These results are consistent with the induction of a cooperative change of the structure of the sugar-binding site or of its immediate vicinity by the ions.     

Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.     

Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase

Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.     

Solution structure of the B-Myb DNA-binding domain: a possible role for conformational instability of the protein in DNA binding and control of gene expression

Double- and triple-resonance heteronuclear NMR spectroscopy have been used to determine the high-resolution solution structure of the minimal B-Myb DNA-binding domain (B-MybR2R3) and to characterize the specific complex formed with a synthetic DNA fragment corresponding to the Myb target site on the Myb-regulated gene tom-1. B-MybR2R3 is shown to consist of two independent protein domains (R2 and R3) joined by a short linker, which have strikingly different tertiary structures despite significant sequence similarities. In addition, the C-terminal region of B-Myb R2 is confirmed to have a poorly defined structure, reflecting the existence of multiple conformations in slow to intermediate exchange. This contrasts with the tertiary structure reported for c-MybR2R3, in which both R2 and R3 have the same fold and the C-terminal region of R2 forms a stable, well-defined helix [Ogata, K., et al. (1995) Nat. Struct. Biol. 2, 309-320]. The NMR data suggest there are extensive contacts between B-MybR2R3 and its DNA target site in the complex and are consistent with a significant conformational change in the protein on binding to DNA, with one possibility being the formation of a stable helix in the C-terminal region of R2. In addition, conformational heterogeneity identified in R2 of B-MybR2R3 bound to the tom-1-A target site may play an important role in the control of gene expression by Myb proteins.     

Factor IX of the blood coagulation system: a review

Factor IX is a factor of the blood coagulation system. Its activation occurs on the surface of phospholipid membranes. It can be activated by the factor VIIa-TF (tissue factor)-Ca2+ complex via an extrinsic pathway and by factor XIa in the presence of Ca2+ via the intrinsic pathway of blood coagulation system activation. The activated factor IXa is a serine proteinase. The main function of the activated factor IXa in complex with factor VIIIa and phospholipids in presence of Ca2+ consists of the activation of factor X. Factor IX is synthesized in the liver and is subject to a number of posttranslational modifications including gamma-carboxylation, beta-hydroxylation, and glycosylation. It forms a subgroup of vitamin K-dependent plasma proteins including factors VII and X and protein C characterized by identical domain structures having high levels of homology. Factor IX consists of an NH2-terminal Gla domain, two epidermal growth factor (EGF)-like domains, and a C-terminal domain containing Ser in its active site. Factor IX deficiency in human plasma results in the disease known as hemophilia B.     

Identification of a factor IX binding site on the third apple domain of activated factor XI

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.     

Fluorescence quenching and time-resolved fluorescence studies on Momordica charantia (bitter gourd) seed lectin

Chemical modification studies implicated tryptophan (Trp) residues in the sugar binding activity of Momordica charantia lectin (MCL) [Mazumdar, T., Gaur, N. & Surolia, A. (1981) Eur. J. Biochem. 113, 463-470]. In the present study, the accessibility and environment of Trp residues in MCL were investigated by intrinsic fluorescence quenching and time-resolved fluorescence. The emission lamda max of native MCL in the absence as well as in the presence of 0.1 M lactose was around 335 nm, which shifted to 365 nm in the presence of 8 M urea, suggesting that the Trp residues which are predominantly buried in the hydrophobic core of the native lectin get exposed to the aqueous environment upon denaturation. At a quencher concentration of 0.5 M, the extent of quenching observed for the native MCL with acrylamide, I- and Cs+ was 46%, 17% and 12%, respectively. In the presence of 0.1 M lactose this quenching was smaller, suggesting that the sugar ligand provides a partial protection to the Trp residues. In time-resolved fluorescence measurements, the decay curves could be fitted well to a biexponential function with the estimated life times 0.92 ns and 4.64 ns for the native protein and 1.15 ns and 5.1 ns in the presence of 0.1 M lactose. All these results are consistent with the involvement of Trp residues in the sugar-binding activity of MCL.     

Membrane topography of the T domain of diphtheria toxin probed with single tryptophan mutants

The membrane insertion and translocation of diphtheria toxin, which is induced in vivo by low pH, is thought to be directed by the hydrophobic alpha-helices of its transmembrane (T) domain. In this study the structure of membrane-associated T domain was examined. Site-directed mutants of the T domain with single Trp residues were prepared at the two naturally occurring positions, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were found to undergo the low-pH-induced conformational change observed with wild-type T domain and insert into model membranes at low pH. The location of Trp residues relative to the lipid bilayer was characterized in model membrane vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 deeply inserted, and residue 355 located at an intermediate depth. Residues 206 and 281 exhibited moderately deep insertion. It was also found, in agreement with our previous study using bimane-labeled protein (Wang et al. (1997) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively shallowly inserted state to a more deeply inserted state when the concentration of the T domain in the membrane is increased or the thickness of the membrane bilayer is decreased. In particular, the depth of residue 355 was found to increase under the conditions giving deeper insertion. In contrast, residue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 insert into the lipid bilayer in both T domain conformations. In addition, Trp depths suggest that even in the shallowly inserted state there is a significant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes. Models for the structure of the membrane-inserted T domain are discussed.     

Tryptophan mutants of troponin C from skeletal muscle--an optical probe of the regulatory domain

We have generated a series of chicken skeletal muscle troponin C mutants to study the conformation of the regulatory domain in the N-terminal half of the molecule. These mutants each contained a single Trp at position 22 (helix A), 52 (linker of helices B and C), or 90 (central helix). Some of these mutants also contained additional mutations to introduce a single Cys at a desired position. The mutants were characterized by molecular graphics and CD and found to have a minimum of structural perturbations when compared with the native structure. They also retained the ability to regulate myofibrillar ATPase activity. The fluorescence of Trp22 was sensitive to Ca2+ binding only to the regulatory sites, whereas Trp52 and Trp90 responded to Ca2+ binding to both the regulatory and the Ca2+/Mg2+ sites. The tryptophan quantum yield (Q) of all Trp22-containing mutants was very high (0.33) in the absence of bound Ca2+, compared to that of L-tryptophan in aqueous solution (0.14). Q decreased 25% upon binding of Ca2+ to the regulatory sites. The quantum yields of Trp52 and Trp90 in apo mutants were close to 0.14. In the presence of bound Ca2+ at the regulatory sites, the quantum yield of Trp52 decreased 16%, whereas that of Trp90 increased 25%. Results from acrylamide quenching of the fluorescence of the three Trp residues indicated that Trp22 was the least exposed and Trp52 was the most exposed, consistent with other spectral data that Trp22 was in a relatively nonpolar environment and Trp52 was in a highly polar environment. The ability of Trp52 and Trp90 to sense Ca2+ binding to sites located at both domains suggests inter-domain communication in the protein. These single Trp TnC mutants provide specific signals for probing Ca2+-induced conformational changes in the regulatory domain.     

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum

The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.     

1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H(+)-ATPase. Conformational properties and lipid-peptide interactions

PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H(+)-ATPase, found to regulate the enzyme activity. To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies. High resolution 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation. Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal. Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions. Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed. Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.     

Spectroscopic properties and stability of the neurotoxic complex. Vipoxin and its components

The neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis is a complex between a toxic basic phospholipase A2 (PLA2) and a non-toxic acidic protein inhibitor (Inh). Tryptophan fluorescence parameters are determined for the complex and for its components. Iodide, caesium and acrylamide are not efficient quenchers of the Vipoxin indole emission. Increased accessibilities of tryptophans to ionic and neutral quenchers are found after the dissociation of the complex. Trp 20 and Trp 31 became more 'exposed' in the separated individuals proteins. The indole rings of the complex are located in a positively charged environment. Inspection of the Vipoxin X-ray model showed that the three tryptophyl side chains are located in the interface region between the enzyme and the inhibitor and are completely 'exposed' in the separated components of the complex. In Vipoxin an efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs. Static quenching with acrylamide is also detected in PLA2 and Inh. The free energy changes deltaG D for the unfolding reactions of Vipoxin, PLA2 and Inh are determined in circular dichroism spectroscopy. The complex formation between the toxic PLA2 and the inhibitor increases deltaG HD2O to 23.5 kJ mol-1.     

Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.     

Role of the J-domain in the cooperation of Hsp40 with Hsp70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.     

Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin

Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (nH = 1.3) and a [Ca2+]0.5 of 1.5 microM, plus one low affinity site with an intrinsic dissociation constant (K'D) of 0.5 mM. CR-22k contains three Ca2+-binding sites with nH of 1.3 and [Ca2+]0.5 of 1.2 microM, plus a low affinity site with K'D of 1 mM. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of alpha-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I-III for CR-22k and I-IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins.     

Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate

A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding [Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-9949]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms. The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts. Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure. When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.     

Interaction of substrate and effector binding sites in the ArsA ATPase

The ars operon of plasmid R773 confers resistance to antimonials and arsenicals in Escherichia coli by encoding an ATP-dependent extrusion system for the oxyanions. The catalytic subunit, the ArsA protein, is an ATPase with two nucleotide binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA ATPase is allosterically activated by tricoordinate binding of As(3+) or Sb(3+) to three cysteine thiolates. Previous measurements suggested that the intrinsic fluorescence of tryptophans might be useful for examining binding of Mg2+ ATP and antimonite. In the present study an increase in intrinsic tryptophan fluorescence was observed upon addition of Mg2+ ATP. This enhancement was reversed by addition of antimonite. The ArsA protein contains four tryptophan residues: Trp159, Trp253, Trp522, and Trp524. The first two were altered to tyrosine residues by site-directed mutagenesis. Cells expressing both the arsAW159Y and arsAW253Y mutations retained resistance to arsenite, and the purified W159Y and W253Y proteins retained ATPase activity. While the intrinsic tryptophan fluorescence of the W253Y protein responded to addition of Mg2+ ATP, intrinsic tryptophan fluorescence in the purified W159Y protein was no longer enhanced by substrate. These results suggest that Trp159 is conformationally coupled to one or both of the nucleotide binding sites and provides a useful probe for the interaction of effector and substrate binding sites.     

Structural analysis of nucleic acids by using fluorescence resonance energy transfer (FRET)

We examined changes in the extent of fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In our system, FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. While, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. It should help us to estimate the suitability of designed functional molecules, such as antisense DNA and RNA and ribozymes, that target to specific sites.