Conformational changes of the insulin receptor upon insulin binding and activation as monitored by fluorescence spectroscopy

We have characterized the changes in intrinsic fluorescence that the insulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, coupled with studies monitoring the energy transfer from insulin receptor tryptophan donors to a fluorescent-labeled insulin, allowed us to conclude that the change in anisotropy is due to a conformational change in the receptor induced by hormone binding. Since insulin association is very fast, the time course also allowed us to estimate the slower rate of formation of this conformationally-altered state. The time course of receptor autophosphorylation was measured under similar conditions and was found to be similar to the ligand-induced anisotropy time course. The simultaneous use of two fluorescent-labeled insulin analogs also allowed us to assess the maximum distance between the two hormones bound to the receptor. Addition of ATP produces a large, seemingly instantaneous increase in anisotropy. Our observation that ATP binds to the insulin receptor in the presence and absence of insulin supports the idea that the conformational change produced by insulin binding increases the rate of autophosphorylation rather than increases ATP affinity. A suggested model for these changes is presented.     

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.     

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Preparation and characterization of the active derivative bovine trypsin-kallikrein inhibitor (Kunitz) with the reactive site lysine-15 -- alanine-16 hydrolyzed

The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.     

Relationship between the ultrastructure and biochemical composition of spores and their resistance to high temperature exposure and chemical agents

The author used the spores of B. cereus and of its two mutants (10id -- defective by spore coats, and No. 3 -- DPA-deficient). The mentioned microbes were subjected to the action of vapour (99 degrees), 5% sodium hydroxide solution at a temperature of 50 degreeC, and of 0.25% sodium hypochlorite solution. On the basis of the survival curves it was revealed that a mutant with defective coats possessed the least resistance to the factors under study. On these grounds a conclusion was drawn on the important portective function of the spore coat, and not simply of the presence of DPA, in the mechanism of thermo- and chemical resistance of spores.     

The use of neamine as a molecular template: identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa by mass spectroscopy

Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3'-phosphotransferase type IIa (APH(3')-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics. Standard peptide mapping techniques failed with this enzyme. A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification. By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6', and 1 and 3 of the antibiotic substrates, respectively. These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.     

Probing the substrate alignment at the active site of 15-lipoxygenases by targeted substrate modification and site-directed mutagenesis. Evidence for an inverse substrate orientation

For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus of the substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenases an inverse head to tail substrate orientation has been proposed. However, recent structure-based sequence alignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation. Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated, and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETE oxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the corresponding data for polyenoic fatty acids. 5S,15S- and 14R, 15S-DiH(P)ETE were identified as major reaction products. (ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reaction specificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectively oxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5-C6 double bond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introduction of a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylation and led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenation by the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leu mutant, 5S- and 8S-lipoxygenations were augmented and 14R, 15S-DiH(P)ETE formation was impaired. (v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation from arachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic data and the alterations in the product specificity are consistent with the concept of an inverse head to tail substrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by 15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with its carboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.     

Binding of peptides in solution by the Escherichia coli chaperone PapD as revealed using an inhibition ELISA and NMR spectroscopy

PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended beta-strands by PapD in aqueous solution. The PapD-peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.     

The localisations in liposomal membranes of the tetrahydrofuran ring moieties of the annonaceous acetogenins, annonacin and sylvaticin, as determined by 1H NMR spectroscopy

We studied 34 apparently healthy children and 2 propositi from kindreds with familial juvenile hyperuricaemic nephropathy (FJHN) - a disorder characterised by early onset, hyperuricaemia, gout, familial renal disease and a similarly low urate clearance relative to glomerular filtration rate (GFR) [fractional excretion of uric acid (FEur) 5.1+/-1.6%] in young men and women. In addition to the propositi, 17 asymptomatic children were hyperuricaemic -- mean plasma urate (368+/-30 micromol/l), twice that of controls (154+/-41 micromol/l). Eight of them had a normal GFR ( > 80 ml/min per 1.73 m2), and 11 renal dysfunction, which was severe in 5. The FEur in the 14 hyperuricaemic children with a GFR > 50 ml/min was 5.0+/-0.5% and in the 5 with a GFR < or =50 ml/min was still low (11.5+/-0.2%) compared with controls (18.4+/-5.1%). The 17 normouricaemic children (185+/-37 micromol/l) had a normal GFR (>80 ml/min) and FEur (14.0+/-5.3%). The results highlight the dominant inheritance, absence of the usual child/adult difference in FEur in FJHN and presence of hyperuricaemia without renal disease in 42% of affected children, but not vice versa. Since early allopurinol treatment may retard progression to end-stage renal failure, screening of all relatives in FJHN kindreds is essential.     

The influence of medium formulation on phosphomonoester and UDP-hexose levels in cultured human colon tumor cells as observed by 31P NMR spectroscopy

High-resolution 31P NMR spectroscopy at 11.7 T was used to examine the influence of medium formulation (medium and serum type, and concentrations of glucose and inositol) on the cellular phosphate metabolism of CX-1 cells, a human colon cancer cell line derived from HT-29 cells. Striking differences in the 31P spectra of harvested CX-1 cells were observed. The largest variation was seen in the phosphocholine and UDP-hexose levels (up to seven-fold changes), with smaller differences in the levels of other phosphate metabolites. The major UDP-hexose species were found to be UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (ca 2:1 ratio), which have been proposed in the literature to be markers of cell differentiation status. Medium-induced alterations in metabolite levels were much greater than the normal variations seen in CX-1 control samples grown under identical conditions. They even exceeded the characteristic differences observed between different human tumor cell lines grown under one set of culture conditions. The remarkable sensitivity of CX-1 cellular phosphate metabolism to the culture environment has implications for the comparison of in vitro vs in vivo spectra, and for the interpretation of effects due to growth and therapy.     

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.     

Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy

The C2A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C2A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C2A was double-labeled with 13C and 15N, and triple-resonance NMR data were collected from C2A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg233 and Phe234 of loop 3 (a loop that also binds Ca2+) and His198, Val205, and Phe206 of loop 2. These residues lie along a straight line on a surface ridge of the C2A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His254; however, because His254 is located on the other side of the molecule from the phospholipid docking site defined by the other residues, its shifts may result from nonspecific interactions. The results show that the "docking ridge" responsible for Ca2+-dependent membrane association is localized on the opposite side of the C2A domain from the transmembrane and C2B domains of synaptotagmin.     

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.     

Relationship of role playing and knowledge of appropriate behavior to assertion in the natural environment.

Role-play tests have become one of the standard ways to assess interpersonal behavior, yet their validity has not been demonstrated. A study compared behavior in a series of role-play situations with behavior in identical in vivo encounters. 28 psychiatric patients (mean age 32.3 yrs) were recruited as Ss. Each was assessed in a series of in vivo situations, in a role-play test, and in a structured interview during which they were asked what someone "should" do in a variety of interpersonal interactions. Results do not support the validity of the role-play procedure. Behavior in the role-play test was not highly related to behavior in the parallel in vivo situations. There also was a greater correspondence between interview responses and in vivo behavior than between interview responses and role play. Alternative hypotheses about this pattern of results are discussed, and it is suggested that the process of role playing might be associated with unique response demands that produce an idiosyncratic response pattern. (36 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Relationship of classroom behavior to the accuracy of the match between material difficulty and student ability.

Examined (a) the level of difficulty of material used for reading instruction with 71 2nd-6th graders and (b) the relationship between the material difficulty/student ability level difference scores and classroom adjustment. Readability formulas determined material difficulty; individual reading tests determined ability levels (the Gray Oral Reading Test, Form B, and the Spache Diagnostic Reading Scales); the Devereux Elementary School Behavior Rating Scale assessed classroom behavior. Ss tended not to receive instruction in material at a level of difficulty equal to their tested ability. Material difficulty/ability level difference scores were significantly related to classroom adjustment: Behavior improved as the material became easier for Ss. Less accurately matched material therefore tended to be related to improved behavior. Unexpected relationships thus exist between classroom behavior and level of difficulty of instructional material. (21 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Estimation of nitrite ions by the formation of aryl nitroanion radicals in the reaction with the aminoarene 3,5-dibromo-4-amino-benzenesulfonic acid by EPR spectroscopy. The use of 15N-nitrite as an internal standard

The reaction between the aminoarene 3,5-dibromo-4-aminobenzenesulfonic acid (Na-salt) and nitrite ions gives rise to the corresponding nitroanion radical. In the reaction the amino group of the parent substance was replaced by NO2 from the nitrite ions. This leads to two different EPR spectra when the reaction was made with 14N and 15N nitrite. Furthermore, the two spectra were completely separated with no overlaps, a property which was utilised for a quantitative evaluation of samples of 14N nitrite by means of the addition of a known amount of 15N nitrite as an internal standard. Variation of the concentration of the nitroanion radicals was eliminated since the variation will affect the 14N and 15N products to the same extent. The amplitudes of the spectral components M1 = +1 of the 14N spectrum and M1 = +1/2 of the 15N spectrum were used for the evaluation of the 14N nitrite content of the sample.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.