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Hydrogenation of 1,3-butadiene and 1-butyne over the Rh/chitosan catalyst in gaseous and liquid phases was investigated by using the NMR and parahydrogen-induced polarization technique. The Rh/chitosan system was a very selective catalyst for the partial hydrogenation of 1,3-butadiene and 1-butyne to 1- and 2-butenes. The catalyst demonstrated high activity in the hydrogenation of 1-butyne after its activation in the hydrogenation of 1,3-butadiene. Therefore, the Rh/chitosan catalyst was used for the catalytic removal of 1,3-butadiene and 1-butyne from gaseous butene streams. Moreover, the syn- and anti-addition of the hydrogen molecule to substrates proceeded in a pairwise manner in the gas phase heterogeneous hydrogenation as was demonstrated by parahydrogen-induced polarization. However, in the liquid phase hydrogenation of 1-butyne, both hydrogen atoms from the same hydrogen molecule were added to 1-butyne only through the syn-addition route. [ABSTRACT FROM AUTHOR]

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We present experimental evidence for a cooperative unfolding transition of an α-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations. [ABSTRACT FROM AUTHOR]

The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces. [ABSTRACT FROM AUTHOR]

State-of-the-art methods based on CNS and CYANA were used to recalculate the nuclear magnetic resonance (NMR) solution structures of 500+ proteins for which coordinates and NMR restraints are available from the Protein Data Bank. Curated restraints were obtained from the BioMagResBank FRED database. Although the original NMR structures were determined by various methods, they all were recalculated by CNS and CYANA and refined subsequently by restrained molecular dynamics (CNS) in a hydrated environment. We present an extensive analysis of the results, in terms of various quality indicators generated by PROCHECK and WHAT_CHECK. On average, the quality indicators for packing and Ramachandran appearance moved one standard deviation closer to the mean of the reference database. The structural quality of the recalculated structures is discussed in relation to various parameters, including number of restraints per residue, NOE completeness and positional root mean square deviation (RMSD). Correlations between pairs of these quality indicators were generally low; for example, there is a weak correlation between the number of restraints per residue and the Ramachandran appearance according to WHAT_CHECK ( r = 0.31). The set of recalculated coordinates constitutes a unified database of protein structures in which potential user- and software-dependent biases have been kept as small as possible. The database can be used by the structural biology community for further development of calculation protocols, validation tools, structure-based statistical approaches and modeling. The RECOORD database of recalculated structures is publicly available from . Proteins 2005. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The homodimeric HU protein from the hyperthermophile Thermotoga maritima (HUTmar) is a model system which can yield insights into the molecular determinants of thermostability in proteins. Unusually for a thermostable protein, HUTmar exists in a structurally heterogeneous state as evidenced by the assignment of two distinct and approximately equally populated forms in solution. Relaxation measurements combined with chemical shift, hydrogen exchange, and nuclear Overhauser enhancement data confirm the main structural features of both forms. In addition, these data support a two-state model for HUTmar in which the major form closely resembles the X-ray structure while the very flexible minor form is less structured. HUTmar may therefore be a new example of the small class of hyperthermostable proteins with unexpected flexibility. [Copyright &y& Elsevier]

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the Zn K edge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess β-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboom et al. (997), Curr. Biol. 7, 739-746; (2000), J. Biomol. NMR, 18, 119-28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations with EXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 Å (Debye-Wallertype factor 0.009 Ų) and two imidazole ligands with the N atoms at 1.99 Å (Debye-Waller-type factor 0.005 Ų). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn-S and Zn-N distances of 2.3 Å and 2.0 Å, respectively). [ABSTRACT FROM AUTHOR]

The lac repressor–operator system is a model system for understanding protein–DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high‐resolution structure of a dimer of the DNA‐binding domain of lac repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48° of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence‐dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor–operator system is unique among other protein–DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction. [ABSTRACT FROM AUTHOR]

The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an α-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right half-sites with different spacer lengths. [ABSTRACT FROM AUTHOR]

¹H-NMR spectra at 600 MHz and 270 MHz and photo-chemically induced dynamic nuclear polarization (photo-CIDNP) spectra at 360 MHz of β-endorphon in the presence of sodium dodecyl sulfate (SDS) micelles are reported and discussed in terms of structural changes and immobilization upon binding. On addition of micelles several NH protons show slow H-D exchange rate in ²H2O at p²H 4.6, 25&degC, which indicates that some regions of the polypeptide are buried and shielded from the direct interaction with the solvent. Moreover, in the methyl region of the spectrum strong changes are detected both in chemical shifts and line-widths, suggesting an appreciable interaction between the hydrophobic residues of β-endorphin and the detergent micelles. [ABSTRACT FROM AUTHOR]

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1–51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360MHz and 500MHz. Individual assignments were obtained at 18 °C for the backbone protons of 44 out of the total of 5l amino acid residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg,-35, Glu-36, Lys-37 and Ile-48. Complete assignments of the non-labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn-46 and Asn-50 the δ amide protons were also identified. The chemical shifts for the assigned resonances at 18 °C are listed for an aqueous solution at pH 4.9 and at pH 6.8. [ABSTRACT FROM AUTHOR]

31P nuclear magnetic resonance spectra of the spectra phototrophic bacterium (Rhodopseudomonas sphaeroides reveal the presence of inorganic phosphate, sugar phosphates and two non-identified P,P¹-diesterified pyrophosphate compounds. Due to the presence of paramagnetic cations the resonances of these compounds can only be detected after repeated washing of the bacterial cells with a buffer, containing EDTA plus excess Mg2+. Washing with Mg2+-free EDTA buffer deteriorates the structural integrity of the membranes of Rps. sphaeroides. This is indicated by the appearance of an extra resonance peak in the spectra of these cells in a region where the phospholipids absorb and by a fivefold increase in proton permeability of the cytoplasmic membrane of Rps. sphaeroides under these conditions. Upon illumination of the cell suspension in the NMR tube the generation of a transmembrane pH gradient can be inferred from the shift in the resonances of extracellular and intracellular inorganic phosphate. Intracellular inorganic phosphate shows one homogeneous resonance peak upon illumination. This demonstrates that the mixing system, which has been developed for this application, functions efficiently. The magnitude of the light-dependent pH difference is 0.8 at an external pH 6. The width at half height of the internal inorganic phosphate peak is essentially independents of internal pH from pH 5-8, remains unchanged upon addition of uncoupler and is inversely proportional to the number of EDTA washings applied. These observations indicate that the inorganic phosphate NMR peak width is predominantly determined by the presence of a residual amount of paramagnetic cations, rather than by a broad distributions of internal pH values over the cells. Ionophores have an effect on the light-dependent pH-gradient in accordance with the cemiosnotic theory; valinomycin increases, and carbonylcyanide p-trifluoromenthoxyphenylhydrazone decreases, the magnitude of this gradient. [ABSTRACT FROM AUTHOR]

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 Å for the backbone atoms and 1.12 A for all atoms for residues 6–36 and 46–67. The structure of IF1 is characterized by a five-stranded β-barrel. The loop connecting strands three and four contains a short 310 helix but this region shows considerably higher flexibility than the β-barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and call be identified as the oligomer-binding motif. Several proteins of this family are nucleic acid-binding proteins. This suggests that IF1 plays its role ill the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes. [ABSTRACT FROM AUTHOR]

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Asn52 of its α subunit (αhCG) is essential for signal transduction, whereas the N-glycan at Asn78 stabilizes the structure of the protein. In this study, an almost complete ¹H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of αhCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of αhCG in solution, which was determined based on NOE data, is partially similar to that of the α subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33–58. continue [ABSTRACT FROM AUTHOR]

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by ¹H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded β-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton-proton distance constraints, 14 hydrogen bond distance constraints and 55 Φ and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09±0.02 nm for backbone atoms (residues 11–30, 37–50, 57–69, 83–89). The protein comprises two identical domains, each containing a three-strand β-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase. [ABSTRACT FROM AUTHOR]

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20- ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin. [ABSTRACT FROM AUTHOR]

One- and two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two-dimensional NOE spectra recorded for the enzyme-inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase-A2-micelle complex: (i) the 38-kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle-binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)-dodecyl-2-aminohexanol-1- phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd ≤ 10 μM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical-shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (< 0.4 nm) near Phe5, Ile9, Phe22 and Phel06. Comparison of our results on the enzyme-inhibitor-micelle ternary complex with the crystal structure of the enzyme-inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. &Verheij, H. M. (1990) Nature 347, 689–691] shows that the mode of inhibitor binding is similar. [ABSTRACT FROM AUTHOR]

Cyclobutane-type photodimers of dinucleoside monophosphates dCpdT, dTpdC and dTpdT were prepared by ultraviolet irradiation in the presence of acetophenone as photosensitizer. The cytosine-containing derivatives were found to deaminate forming uracil products. Using one-and two-dimensional NMR, the photoproducts were characterized as cis-syn and trans-syn cyclobutane photodimers. On the basis of NOE data the structures of the cis-syn and trans-syn products of dUpdT were determined using distance-geometry and restrained-energy- minimization methods. The cis-syn structures showed (high-ANTI/SYN)/high-ANTI glycosidic linkages while the trans-syn structures were in the SYN-ANTI region. The backbone conformations of both structures were in fair agreement with the coupling-constant-data. The trans-syn structures were found to be very rigid and similar in all three products. For the three cis-syn structures more conformational freedom and more variation among the three structures was observed. [ABSTRACT FROM AUTHOR]

A complex between the headpiece amino-terminal residues 1-56 of lac repressor (HP56) and an 11-bp lac operator fragment was studied by ¹H NMR. The sequence specific assignment of the exchangeable and non-exchangeable protons has been accomplished. Several protons have favourable chemical shifts in the complex, therefore new intraprotein NOEs could be found that had not been unambiguously identified in the free protein. By comparison, most of these intraprotein NOEs are also present in the spectra of the free headpiece but some are different. Furthermore, several new protein DNA NOEs could be identified. The NOE between the side-chain amide protons of Gln18 and C5H of C7 confirms the specific contact between these residues which was proposed from genetic experiments [Ebright, R. M. (1985) J. Biomol. Struct. & Dyn. 3, 281-297]. The implications of the new data for the interaction between the lac repressor headpiece and its operator are discussed. [ABSTRACT FROM AUTHOR]

The shortened analogue of insulin, des-(B26-B30)-pentapeptide insulin, has been characterized by twodimensional ¹H NM R. The ¹H resonance assignments and the secondary structure in water solution are discussed. The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1. [ABSTRACT FROM AUTHOR]

The secondary structure of crambin in solution has been determined using two-dimensional NMR and is found to be essentially identical to that of the crystal structure. The H - D exchange of most amide protons can be accounted for in terms of the hydrogen bonds found in the X-ray structure. Exceptions are the amide protons of Cys-4 and Ser-6, which exchange more slowly than expected, and of Asn-46 for which the exchange is faster. These results might be explained by a slightly different conformation of the C-terminal region of the protein in solution. The slow exchange of the amides of Cys-32 and Glu-23 might be due to aggregation involving an extremely hydrophobic part of the protein in solution. [ABSTRACT FROM AUTHOR]

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCG&TTcirc;GCG) · d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the IH chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer. [ABSTRACT FROM AUTHOR]

Reports on the recording of one-dimensional and two-dimensional photochemically-induced dynamic nuclear polarization nuclear magnetic resonance spectra for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. Accessibility of all three Trp residues to the flavin dye in anthopleurin and toxin II; Examination of the pH dependences of the photo-CIDNP spectra of all three polypeptides.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms. [ABSTRACT FROM AUTHOR]

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galecfin-1/chicken galectins and of Trp194 in marine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type routine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL in assumed to provide information on the role of a certain residue for functional aspects. When single site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl tings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins. [ABSTRACT FROM AUTHOR]

The photochemically-induced dynamic nuclear polarization technique has been used to investigate the access of a photoexcited flavin dye to tyrosyl and histidyl residues in [Met]enkephalin and human and camel β-endorphins, both alone and in the presence of n-dodecylphosphorylcholine micelles. The results indicate that the mode of binding of Tyr-1, but not of residue 27, is similar in the two endorphins and differs from that of Tyr-1 in [Met]enkephalin. In human β-endorphin, accessibility and mobility of Tyr-27 are strongly reduced in tile presence of lipid at physiological pH, whereas in camel β-endorphin His-27 becomes immobilized only at high pH. Moreover, nuclear Overhauser enhancement experiments suggest a rigidifying influence of the peptide on the polar head groups of the micelles. [ABSTRACT FROM AUTHOR]

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded amino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few amino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form. [ABSTRACT FROM AUTHOR]

Bacteriorhodopsin has been reconstituted into egg-phosphatidylcholine vesicles by various methods. The resulting preparations have been analyzed on density gradients and by freeze-fracture electron microscopy. The homogeneity of the vesicle preparations and the light-induced intravesicular pH changes have been studied by 31P-NMR, using glucose 6-phosphate as pH probe. It is concluded that bacteriorhodopsin is incorporated in the inside-out mode in vesicles up to about 100 nm. Above this diameter, more or less random insertion takes place. [ABSTRACT FROM AUTHOR]

Porcine pancreatic colipase possesses a particular three-dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343-354; J. Wieloch, B. Borgström, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622-1628]. The domain corresponds to the 49-57 and 77-86 β-sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29-42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo-CIDNP) studies of the complexes of colipase with organized bile salt micelles. The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr-I and Tyr-II (Tyr-56 and Tyr-57) and His-II (His-86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo-CIDNP effects of Tyr-I and Tyr-II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219-222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo-CIDNP effects observed in the colipase-micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic. A general model for micelle binding to colipase is proposed and involves a two-step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase. [ABSTRACT FROM AUTHOR]

1. The aromatic proton resonances in the 360-MHz ¹H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessible to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180° about their Cβ−Cγbond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues. [ABSTRACT FROM AUTHOR]

Review: 105 refs. [ABSTRACT FROM AUTHOR]