Zur Kenntnis des Faktors F430 aus methanogenen Bakterien: Struktur des porphinoiden Ligandsystems

Factor F430 from Methanogenic Bacteria: Structure of the Porphninoid Ligand System A structure is proposed for F430M, a non-cristalline methanolysis product of isolates of the nickel-containing, porphinoid factor F430 from Methanobacterium thermoautotrophicum. Crucial to the structure determination are five incorporation experiments with M. thermoautotrophicum (strain Marburg) in which the specifically mono-¹³C-labeled biosynthetic precursors (2-¹³C), (3-¹³C), (4-¹³C)-, (5-¹³C) ALA (ALA = δ-amino-levulinic acid) and L-(methyl-¹³C)methionine were incorporated into F430 with high efficiency. The ¹³C-NMR,-spectra of the specifically labeled F430M samples derived therefrom, together with the UV./VIS. spectral data of F430M, contain all the information necessary for the deduction of the constitution of the F430M chromophore, assuming the established pattern of porphinoid biosynthesis to be operative in F430 biosynthesis. ¹H-NMR. spectroscopy and, in particular, ¹H-NMR.-NOE-difference spectroscopy corroborates and completes the constitutional assignments and, furthermore, makes possible an almost complete derivation of the molecule's relative configuration. Schemes 3 and 4 summarize the results of ¹H-NMR. spectroscopy, presenting them within the context of the proposed structure for F430M. The assignment of absolute configuration implied in the formula is given preference because of F430M's very close structural and (assumed) biosynthetic relationship to sirohydrochlorin and vitamin B₁₂ (with respect to ring C, the assignment is based on degradative evidence). According to the proposed structure, the nickel complex F430M possesses an uroporphinoid (Type III) ligand skeleton with an additional carbocyclic ring and a chromophore system not previously encountered among natural porphinoids. It can be considered to be a (tetrahydro) derivative of the corphin system, combining structural elements of both porphyrins and corrins.     

Zur Kenntnis des Faktors F430 aus methanogenen Bakterien: Über die Natur der Isolierungsartefakte von F430, ein Beitrag zur Chemie von F430 und zur konformationellen Stereochemie der Ligandperipherie von hydroporphinoiden Nickel(II)-Komplexen

Factor F430 from Methanogenic Bacteria: On the Nature of the Isolation Artefacts of F430, a Contribution to the Chemistry of F430 and the Conformational Stereochemistry of the Ligand Periphery of Hydroporphinoid Nickel(II) Complexes Factor F430 ( 1 ), a coenzyme from methanogenic bacteria, when heated in aqueous solution isomerizes to 12,13-di-epi-F430 ( 5 ) via 13-epi-F430 ( 3 ). The equilibrium mixture of the three F430 isomers in aqueous phosphate buffer solution (pH 7, 100°) contains 88 % of 5 , 8 % of 3 , and 4 % of 1 (Scheme 1). The structural assignment for the F430 isomers rests on FAB-MS-, UV/VIS-, ¹H- and ¹³C-NMR spectra of their pentamethyl esters. Chemical proof for the double epimerization at the two chiral centers of F430's ring C was provided by ozonolytic degradation of the di-epimer to give a ring-C-derived succinimide derivative that was shown to be the enantiomer of the one previously obtained by ozonolysis of F430M (see Scheme 2). The two F430 ring-C epimers 3 and 5 are the isolation artefacts described in the earlier F430 literature. F430 is susceptible to autoxidation in air and the product, that absorbs at 560 nm, was shown to be the 12,13-didehydro derivative 8 of F430 by spectroscopic characterization of its pentamethyl ester 9 . The dehydrogenation product 8 can be diastereoselectively reduced with Zn in AcOH to give natural F430 as the main product rather than the thermodynamically more stable F430-di-epimer (Scheme 3). In the double epimerization of F430, the two ring-C side chains change from a trans-quasi-diaxial arrangement to the (locally) enantiomorphic position in which the same side chains are again in a trans-quasi-diaxial arrangement. This equilibrium paradox as well as the kinetic diastereoselectivity of the reduction of 12,13-didehydro-F430 ( 8 ) are rationalized to be consequences of the general phenomenon documented earlier (see the preceding paper) according to which hydroporphinoid Ni(II) complexes all show a characteristic conformational ruffling of their ligand system due to the tendency of the (small) Ni(II) ion to contract the size of the ligand's central coordination hole (see Fig. 5 and 6).     

Zur Kenntnis des Faktors F430 aus methanogenen Bakterien: Struktur des proteinfreien Faktors

Factor F430 from Methanogenic Bacteria: Structure of the Protein-free Factor Factor F430, the porphinoid nickel-containing coenzyme of the methylcoenzyme-M reductase of metanogenic bacteria is shown to be the 3³,8³,12²,13³,18²-pentaacid derivative of the pentamethylester F430M, the structure of which had been determined previously (see structural formulae 1 and 2 ). The structure assignment rests on chromatographic, UV/VIS-, CD-, IR-, and ¹³C-NMR-spectroscopic as well as FAB-mass spectral comparision of F430 with F430M and the pentaacid prepared by acid-catalyzed hydrolysis of F430M. In the cells of Methanobacterium thermoautotrophicum, factor F430 is present in a ‘bound’ and also, depending on the growth conditions, in ‘free’ form, the latter being defined as the part of total F430 that can be extracted from the cells under extremely mild conditions (80% EtOH at 0–4°). From the (protein)-‘bound’ form, F430 is extracted by subsequently treating the cells at 0–4° with 80% EtOH containing (e.g.), 2m LiCi. From both sources, the extracted factor is the same pentaacid, and there is no indication for the existence of a protein-free F430 species that would contain additional (covalently bound) structural elements.     

Coenzyme F430 from Methanogenic Bacteria: Complete Assignment of Configuration Based on an X-Ray Analysis of 12,13-Diepi-F430 Pentamethyl Ester and on NMR Spectroscopy

The structure of a derivative of coenzyme F430 from methanogenic bacteria, the bromide salt of 12,13-diepi-F430 pentamethyl ester ( 5 , X = Br), was determined by X-ray structure analysis. It reveals a more pronounced saddle-shaped out-of-plane deformation of the macrocycle than any hydroporphinoid Ni complex investigated so far. The crystal structure confirms the constitution proposed for coenzyme F430 ( 2 ) and shows that in the epimer 5 , the three stereogenic centers in ring D, C(17), C(18), and C(19), have the (17S)-, (18S)-, and (19R)-configuration, respectively. Deuteration and 2D-NMR studies independently demonstrate that native coenzyme F430 (2) has the same configuration in ring D as the epimer 5 . Therefore, our original tentative assignment of configuration at C(19) and C(18) [1] has to be reversed. This completes the assignment of configuration for all stereogenic centers in coenzyme F430, which has the structure shown in Formula 2 .     

Herstellung und Eigenschaften einiger hydrocorphinoider Nickel(II)-Komplexe

Preparation and Properties of some Hydrocorphinoid Nickel(II)-Complexes Corphin derivatives synthesized earlier in our laboratory have been used for the preparation of tetrahydro- and hexahydrocorphinoid nickel(II)-complexes containing novel chromophore systems. The UV./VIS. and ¹H- and ¹³C-NMR. spectral data of these complexes were relevant to the structure determination of Factor F430 described in the following paper.     

Derivatives of Coenzyme F430 with a Covalently Attached α‐Axial Ligand. Part II

Coenzyme F430 pentamethyl ester 2 was partially hydrolyzed to a mixture of the five F430 tetramethyl esters 7 – 11 , which were separated by HPLC and identified by means of a full NMR characterization. The tetramethyl ester with a free COOH group at the side chain at C(3) of F430 was coupled to the N‐terminus of the peptidic spacer? ligand construct 12 selected and studied as described before. The UV/VIS and NMR spectra in CH₂Cl₂/3,3,3‐trifluoroethanol 6 : 1 show that the new derivative, the Ni^II(3³‐dehydroxy‐8³,12²,13³,18²‐tetra‐O‐methyl‐F430‐3³‐yl)‐L ‐prolyl‐L ‐prolyl‐N^π‐methyl‐L ‐histidine methyl ester ( 13 ), is an intramolecular, pentacoordinate, paramagnetic complex. In the same solvent system, the parent 3³,8³,12²,13³,18²‐penta‐O‐methyl‐F430 ( 2 ) is four coordinate and diamagnetic even in the presence of equimolar 1H‐imidazole. Protonation of the axially coordinating histidine residue of 13 gave the diamagnetic tetracoordinate base‐off form, which allowed us to establish the constitution of 13 by NMR.     

Coenzyme F430 from Methanogenic Bacteria: Mechanistic studies on the reductive cleavage of sulfonium ions catalyzed by F430 pentamethyl ester

Mechanistic questions regarding the reductive cleavage of sulfonium ions by the Ni^I form of coenzyme F430 pentamethyl ester (F430M) were addressed in a series of kinetic studies and isotope labeling experiments. In neat DMF, methane formation from dialkyl(methyl)sulfonium ions consistently showed a delay time of ca. 1 h. In the presence of excess propanethiol, no delay was observed and methane formation followed pseudo-first-order kinetics with a logarithmic dependence of the initial rate on the concentration of propanethiol. From the temperature dependence of the reaction rate, an estimate for the activation parameters of ΔH^# = 49 kJ mol^?1 and (apparent) ΔS# = –114 J K^?1 mol^?1 was derived. The observation of deuterium incorporation into methane from (CH₃)₂CHOD, but not from (CH₃)₂CDOH, indicates that the fourth H-entity is introduced into CH₄ as a proton, and that free CH₃ radicals are not involved. In contrast to the reaction with the homogeneous one-electron reductant sodium naphthalide, the F430M-catalyzed reduction of mixed dialkyl(methyl)sulfonium ions showed a pronounced selectivity for the cleavage of Me? S over that of alkyl-S (alkyl ≠ Me) bonds. Mechanisms that are consistent with these results, as well as possible explanations for the time delay and the apparent highly negative entropy of activation, are discussed.     

Coenzyme F430 from Methanogenic Bacteria: Detection of a Paramagnetic Methylnickel(II) Derivative of the Pentamethyl Ester by 2H-NMR Spectroscopy

A methylnickel(II) derivative of coenzyme F430 ( 1 ) was proposed as an intermediate in the enzymic process catalyzed by methyl-CoM reductasc. Indirect evidence points to formation of CH₃–F430M^II in the reaction of F30M¹ (obtained from F430M^II ( 2 )) with eleclrophilic methyl donors. The results presented here show, that such a compound does exist. A paramagnetic CD₃–Ni^II derivative 5b of the pentamethyl ester 2 (F430M) of coenzyme F430 was prepared by in situ methylation with (CD₃)₂Mg and characterized by its isotropically shifted ²H-NMR spectrum. At ?40°, the very broad D-signal of the axially coordinated CD₃ group is found at ?490 ppm. Comparison with the ²H- and ¹H-NMR spectra of mcthyl(tetramethylcyclam)nickel(II) derivatives 4 ([Ni^II(CH₃))(tmc)]CF₃SO₃ ( 4a ) is the only isolated CH₃–Ni derivative of a N₄macrocyclic Ni^II complex' shows that the large isotropic shift to high field is characteristic for a Me group axially bound to the Ni center. The temperature dependence of the isotropic shift of the CD₃–Ni group in both 4b and 5b follows Curie's law and yields ²H hyperfine coupling constants of ?0.65 ( 4b ) and ?0.85 MHz ( 5b ), respectively. The ¹H-NMR spectrum indicates that, in contrast to the five-coordinate monochloro complex [Ni^IICl(tmc)]⁺, intermolecular exchange of the axial ligand in [Ni^II(CH₃)(tmc)]⁺ 4a is either slow at the NMR time scale or does not occur at all.     

Derivatives of Coenzyme F430 with a Covalently Attached α‐Axial Ligand. Part I

X‐Ray structures of the enzyme methyl‐coenzyme M reductase show that the Ni‐center in the prosthetic group coenzyme F430 is penta‐ or hexacoordinated with the carboxamide group of a glutamine residue occupying the axial coordination site on the α‐side of the macrocycle. To obtain diastereoselectively coordinated complexes for mechanistic and spectroscopic studies of the free coenzyme in solution, we aimed to prepare partial‐synthetic derivatives of coenzyme F430 that have a coordinating group attached via a linker to one of the propanoic acid side chains. By using molecular‐mechanics calculations and two different conformational search methods, a set of 50 structures containing imidazole or pyridine units as potential ligands were computationally tested according to geometric criteria defining coordinating conformations. The best candidates proved to be proline‐containing tri‐ and tetrapeptides with a methyl‐histidine as the C‐terminal residue. These linkers were synthesized, and their conformation was determined by NMR. Refinement of the molecular modeling by using the experimentally determined geometric restraints allowed us to decide that the tripeptide Pro‐Pro‐His(π‐Me)‐OMe ( 10 ) was the most promising of all tested structures for attachment to the side chain at C(3) or C(13) of F430.     

Direct determination of the number of electrons needed to reduce coenzyme F430 pentamethyl ester to the Ni(I) species exhibiting the electron paramagnetic resonance and ultraviolet-visible spectra characteristic for the MCR(red1) state of methyl-coenzyme M reductase

The UV-visible and electron paramagnetic resonance (EPR) spectra of MCR(red1), the catalytically active state of methyl-coenzyme M reductase, are almost identical to those observed when free coenzyme F430 or its pentamethyl ester (F430M) are reduced to the Ni(I) valence state. Investigations and proposals concerning the catalytic mechanism of MCR were therefore based on MCR(red1) containing Ni(I)F430 until, in a recent report, Tang et al. (J. Am. Chem. Soc. 2002, 124, 13242) interpreted their resonance Raman data and titration experiments as indicating that, in MCR(red1), coenzyme F430 is not only reduced at the nickel center but at one of the C=N double bonds of the hydrocorphinoid macrocycle as well. To resolve this contradiction, we have investigated the stoichiometry of the reduction of coenzyme F430 pentamethyl ester (F430M) by three independent methods. Spectroelectrochemistry showed clean reduction to a single product that exhibits the UV-vis spectrum typical for MCR(red1). In three bulk electrolysis experiments, 0.96 +/- 0.1 F/mol was required to generate the reduced species. Reduction with decamethylcobaltocene in tetrahydrofuran (THF) consumed 1 mol of (Cp)(2)Co/mol of F430M, and the stoichiometry of the reoxidation of the reduced form with the two-electron oxidant methylene blue was 0.46 +/- 0.05 mol of methylene blue/mol of reduced F430M. These experiments demonstrate that the reduction of coenzyme F430M to the species having almost identical UV-vis and EPR spectra as MCR(red1) is a one-electron process and therefore inconsistent with a reduction of the macrocycle chromophore.     

The nickel-sirohydrochlorin formation mechanism of the ancestral class II chelatase CfbA in coenzyme F430 biosynthesis

The class II chelatase CfbA catalyzes Ni²⁺ insertion into sirohydrochlorin (SHC) to yield the product nickel-sirohydrochlorin (Ni-SHC) during coenzyme F430 biosynthesis. CfbA is an important ancestor of all the class II chelatase family of enzymes, including SirB and CbiK/CbiX, functioning not only as a nickel-chelatase, but also as a cobalt-chelatase in vitro. Thus, CfbA is a key enzyme in terms of diversity and evolution of the chelatases catalyzing formation of metal-SHC-type of cofactors. However, the reaction mechanism of CfbA with Ni²⁺ and Co²⁺ remains elusive. To understand the structural basis of the underlying mechanisms and evolutionary aspects of the class II chelatases, X-ray crystal structures of Methanocaldococcus jannaschii wild-type CfbA with various ligands, including SHC, Ni²⁺, Ni-SHC, and Co²⁺ were determined. Further, X-ray crystallographic snapshot analysis captured a unique Ni²⁺-SHC-His intermediate complex and Co-SHC-bound CfbA, which resulted from a more rapid chelatase reaction for Co²⁺ than Ni²⁺. Meanwhile, an in vitro activity assay confirmed the different reaction rates for Ni²⁺ and Co²⁺ by CfbA. Based on these structural and functional analyses, the following substrate-SHC-assisted Ni²⁺ insertion catalytic mechanism was proposed: Ni²⁺ insertion to SHC is promoted by the support of an acetate side chain of SHC.

The substrate-assisted nickel chelatase mechanism of CfbA in coenzyme F430 biosynthesis was unveiled by X-ray crystal structure analysis.     

Über hochverzweigte aliphatische Verbindungen

Zusammenfassung Die Konstitution des Triisopropylamins wird durch Vergleich mit den bisher zu wenig beschriebenen Isomeren gestützt. Die Umsetzung von Dialkylformamiden mit Grignardverbindungen wird zur Charakterisierung der Dialkylformamide vorgeschlagen; es wird gezeigt, daß durch Anwendung eines Gemisches von RMgX mit RMgX entsprechend unsymmetrisch gebaute tertiäre Amine R₂N–CHRR entstehen.Herrn Prof. Dr.O. Kratky zum 60 Geburtstag gewidmet.a) 1.Mitt.:F. Kuffner undE. Polke, Mh. Chem.82, 330 (1951); b) 2. Mitt.:F. Kuffner undW. Seifried, ebenda Mh. Chem.83, 748 (1952).     

Porphyrins 42. Ground and excited state calculations on the isomers of free base porphine and sirohydrochlorin

Symmetry instabilities were encountered during MINDO/3 geometry optimizations of the sirohydrochlorin and porphine isomers leading to bond alternating optimal structures. Transition energies and oscillator strengths were calculated with INDO/S/CI. Our calculations predict the ground state cis and trans isomers of sirohydrochlorin to be close in energy and confirm the experimental assignment of the absorptions bands, with the cis tautomer having a red shifted spectrum.Part 41. Gouterman, M., Sayer, P., Shankland, E., Smith, J. P.: Inorg. Chem. 20, 87 (1981)     

Single-Benzene-Based Solvatochromic Chromophores: Color-Tunable and Bright Fluorescence in the Solid and Solution States

The search for structurally simple chromophores with superior fluorescence brightness and a wide range of solvent compatibility is highly desirable. Herein, a new type of single-benzene-based solvatochromic chromophore with a symmetric bifunctional structure, in which azetidine and ethoxycarbonyl moieties serve as the electron-donating and -withdrawing groups, respectively, is reported. This chromophore exhibits an extraordinary wide range of solvent compatibility and preserves excellent fluorescence quantum yields from nonpolar n-hexane to polar methanol and even in water. Unusually, the symmetric structure of the chromophore shows a distinct color change from bright green to red with increasing solvent polarity and possesses large Stokes shifts (λ=132–207 nm) in the tested solvents. Moreover, this single-benzene-based chromophore displays good photochemical stability in both solution and solid states, and even exhibits reversible mechanochromic luminescence.     

Vinyl monomers bearing chromophore moieties and their polymers. VIII. synthesis and fluorescence behavior of a vinyloxy monomer having an electron‐accepting chromophore moiety,p‐((vinyloxy)methyl)benzonitrile,and its polymers

A vinyloxy monomer having an electron‐accepting chromophore moiety, p‐((vinyloxy)methyl)benzonitrile (VOMBN), was synthesized by reaction of p‐(hydroxymethyl)benzonitrile with ethyl vinyl ether (EVE) in the presence of mercuric acetate. VOMBN can easily be cationically homopolymerized and copolymerized with EVE by using Lewis acids such as boron trifluoride etherate (BF₃ · OEt₂) as a catalyst and radically copolymerized with maleic anhydride (MAn) using AIBN as an initiator. The fluorescence behaviors of VOMBN, its copolymer P(VOMBN‐co‐MAn), and its saturated model compound p‐(ethoxymethyl)benzonitrile (EOMBN) were investigated in acetonitrile. It has been found that the fluorescence intensity of VOMBN is much lower than its copolymer and EOMBN at the same chromophore concentration. A fluorescence “structural self‐quenching effect” (SSQE) is also observed for VOMBN as we have reported previously [Li, F. M.; Chen, S. J.; Li, Z. C.; Qiu, J. J Polym Sci Polym Chem 1996, 34, 1881]. This phenomenon has been attributed to the inter‐ and intramolecular charge transfer interaction between the electron‐accepting cyanophenyl chromophore and the electron‐donating vinyloxy group in the same molecule. The dependence of the fluorescence intensity of VOMBN on solvents of different viscosities is evidence that the SSQE of VOMBN mainly occurs intramolecularly. The fluorescence of EOMBN and P(VOMBN‐co‐MAn) was quenched by a series of electron‐rich vinyloxy compounds which do not have chromophore moieties, such as dihydrofuran (2H‐furan), dihydropyran (2H‐pyran), furan, and EVE. It is observed that the higher the electron‐donating ability of the quenchers, the greater the quenching efficiency. P(VOMBN) and the random copolymers of VOMBN with EVE show broader fluorescence spectra as compared to the alternating copolymer P(VOMBN‐co‐MAn). This indicates that there is a mutual interaction between the adjacent cyanophenyl groups in P(VOMBN) and P(VOMBN‐co‐EVE), whereas such an interaction does not exist for P(VOMBN‐co‐MAn) in which the cyanophenyl groups are isolated by the rigid succinic anhydride rings. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 179–187, 1999     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

Manipulation of Single‐Stranded DNA by Using an Artificial Site‐Selective DNA Cutter Composed of Cerium(IV)/EDTA and Phosphonate–Oligonucleotide Conjugates

An artificial site‐selective DNA cutter to hydrolyze single‐stranded DNA at a desired site was prepared from Ce^IV/ethylenediamintetraacetic acid (EDTA) and two ethylenediamine‐N,N,N′,N′‐tetrakis(methylenephosphonic acid)–oligonucleotide conjugates. By using this cutter, the sense strand of a blue fluorescent protein (BFP) gene was selectively cut at a predetermined site in the chromophore‐coding region. The upstream fragment obtained by the site‐selective scission was ligated with the downstream fragment of the closely related green fluorescent protein (GFP) gene so that the 5′‐ and 3′‐end portions of the chromophore came from the BFP fragment and the GFP fragment, respectively. The recombinant gene was successfully expressed in E. coli and the chimeric chromophore emitted green fluorescence as expected.     

Syntheses of bile pigments. Part 15. First unequivocal assignment of the absolute configuration of an urobilinoid bile pigment by X-ray diffraction analysis of its synthetic precursor

(?)-(4S,16S)-8, 12-bis[de(2-carboxyethyl)]mesourobilin-IIIα hydrochloride ( 8 ) has been synthesized from the enantiomerically pure 1,4,5,10-tetrahydro-1-oxodipyrrin-9-carboxylic-acid precursor 6a whose absolute configuration was determined by X-ray diffraction analysis of the N-[(S)-1-(1-naphthyl)ethyl] carboxamide 7b . The present results prove unequivocally that an (S,S)-configurated urobilin chromophore displays a negative Cotton effect in the VIS absorption range. However, the helicity of the inherently dissymmetric chromophore remains undetermined.     

Origins of the Intermediate Spectral Form in M100 Mutants of Photoactive Yellow Protein

Numerous single‐site mutants of photoactive yellow protein (PYP) from Halorhodospira halophila and as well as PYP homologs from other species exhibit a shoulder on the short wavelength side of the absorbance maximum in their dark‐adapted states. The structural basis for the occurrence of this shoulder, called the “intermediate spectral form,” has only been investigated in detail for the Y42F mutation. Here we explore the structural basis for occurrence of the intermediate spectral form in a M121E derivative of a circularly permuted H. halophila PYP (M121E‐cPYP). The M121 site in M121E‐cPYP corresponds to the M100 site in wild‐type H. halophila PYP. High‐resolution NMR measurements with a salt‐tolerant cryoprobe enabled identification of those residues directly affected by increasing concentrations of ammonium chloride, a salt that greatly enhances the fraction of the intermediate spectra form. Residues in the surface loop containing the M121E (M100E) mutation were found to be affected by ammonium chloride as well as a discrete set of residues that link this surface loop to the buried hydroxyl group of the chromophore via a hydrogen bond network. Localized changes in the conformational dynamics of a surface loop can thereby produce structural rearrangements near the buried hydroxyl group chromophore while leaving the large majority of residues in the protein unaffected.     

Synthesis and structural characterizations of [chromophore]+‐saponite/polyurethane nanocomposites

In this study, three chromophores—p‐nitroaniline, 4‐(4‐nitrophenylazo)aniline, and 4‐[(E)‐2‐{4‐[(E)‐2‐(4‐nitrophenyl)‐1‐diazenyl]phenyl}‐1‐diazenyl]aniline—were intercalated into layered aluminosilicate saponite and then dispersed into the polyurethanes matrix. The intercalated chromophore/saponite complexes were examined by inductively coupled plasma emission and element analysis technologies. The molecular orbital package computation simulation and X‐ray diffraction (XRD) analysis showed that possible configurations of chromophore ions on the gallery surfaces of saponite suggest that the chromophore molecules lie parallel to the basal planes of silicate as an inclined paraffin structure or as pseudo‐multilayers. The XRD and transmission electron microscopy analysis indicated that the delamination of organoclay in the polyurethanes matrix exhibited nanolayers, exfoliated structure, or both. In particular, even at high doping levels up to 15 wt % of organoclay, the [chromophore]⁺‐saponite/polyurethanes film did not display a macroscopic aggregation of layered silicates and showed high transparency. The thermal stability of chromophore was significantly enhanced as intercalated into the layered aluminosilicate saponite, and the glass‐transition temperature of [chromophore]⁺‐saponite/polyurethanes nanocomposites proportionally increased with increased clay content. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1690–1703, 2002