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PubMed1995

Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa.

BACKGROUND: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (-330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin. RESULTS: The crystal structure of the oxidized form of PsCCP has been determined to 2.4 A resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands. CONCLUSIONS: The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce a new residues into the haem pocket.

Amino Acid Sequence↗
PubMed1997

N-terminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.

BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.

Amino Acid Sequence↗
PubMed1992

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Dioxide↗
PubMed1986

1H NMR investigation of cytochrome cd1: complexes with electron-donor proteins.

Mixtures of the dissimilatory nitrite reductase cytochrome cd1 from Pseudomonas aeruginosa and potential electron-donating proteins were prepared in both fully oxidized and fully reduced states and examined by 1H NMR spectroscopy. The relatively narrower lines of the donor proteins enabled them to be clearly observed in spectra in the presence of significant amounts of the high molecular weight cd1. Mixtures of the physiological donor (Pseudomonas ferrocytochrome c-551) and ferrocytochrome cd1 showed specific line-broadening effects on the resonances of c-551 that depended on the mole ratio of c-551 to cd1. The experimental broadening fit a model in which c-551 is in intermediate or fast exchange between free solution and a complex with cd1, with an association constant for the complex in excess of 10(4) M-1. The model yields a minimum estimate for the forward bimolecular rate constant of 5 X 10(7) M-1 s-1 and suggests that the actual value may be much larger. The complexation was independent of pH in the range of 6-8, was independent of ionic strength over a salt concentration range of 20-1000 mM, and possessed a low thermal activation barrier. Mixtures of ferricytochrome c-551 and ferricytochrome cd1 showed no observable NMR perturbations, indicating that any hypothetical complex involving the oxidized forms must follow different dynamical and/or equilibrium conditions. No observable NMR perturbations existed in spectra of mixtures of cd1 and mammalian cytochrome c or Pseudomonas azurin in either oxidation state.

Animals↗
PubMed1986

Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin.

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.

Binding Sites↗
PubMed1988

Quenching of room temperature protein phosphorescence by added small molecules.

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Luminescent Measurements↗
PubMed1976

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.

Acrylamides↗
PubMed2002

Nitrosocyanin, a red cupredoxin-like protein from Nitrosomonas europaea.

Nitrosocyanin (NC), a soluble, red Cu protein isolated from the ammonia-oxidizing autotrophic bacterium Nitrosomonas europaea, is shown to be a homo-oligomer of 12 kDa Cu-containing monomers. Oligonucleotides based on the amino acid sequence of the N-terminus and of the C-terminal tryptic peptide were used to sequence the gene by PCR. The translated protein sequence was significantly homologous with the mononuclear cupredoxins such as plastocyanin, azurin, or rusticyanin, the type 1 copper-binding region of nitrite reductase, and the binuclear CuA binding region of N(2)O reductase or cytochrome oxidase. The gene for NC contains a leader sequence indicating a periplasmic location. Optical bands for the red Cu center at 280, 390, 500, and 720 nm have extinction coefficients of 13.9, 7.0, 2.2, and 0.9 mM(-1), respectively. The reduction potential of NC (85 mV vs SHE) is much lower than those for known cupredoxins. Sequence alignments with homologous blue copper proteins suggested copper ligation by Cys95, His98, His103, and Glu60. Ligation by these residues (and a water), a trimeric protein structure, and a cupredoxin beta-barrel fold have been established by X-ray crystallography of the protein [Lieberman, R. L., Arciero, D. M., Hooper, A. B., and Rosenzweig, A. C. (2001) Biochemistry 40, 5674-5681]. EPR spectra of the red copper center indicated a Cu(II) species with a g(parallel) of 2.25 and an A(parallel) of 13.8 mT (144 x 10(-4) cm(-1)), typical of Cu in a type 2 copper environment. NC is the first example of a type 2 copper center in a cupredoxin fold. The open coordination site and type 2 copper suggest a possible catalytic rather than electron transfer function.

Amino Acid Sequence↗
PubMed2003

Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen.

The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins.

Binding Sites↗
PubMed1999

H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin.

Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.

Asparagine↗
PubMed1999

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

Electrochemistry↗
PubMed2001

Methylation of (2-methylethanethiol-bis-3,5-dimethylpyrazolyl)methane zinc complexes and coordination of the resulting thioether: relevance to zinc-containing alkyl transfer enzymes.

A series of zinc complexes using a new tripodal, N(2)S, heteroscorpionate ligand (L3SH) that is isostructural and isoelectronic with the well-known N(3) trispyrazolylborates have been methylated in solution and the coordination properties of the resulting thioether examined. This system models the reactivity of zinc-containing enzymes involved in alkyl group transfers such as the DNA repair protein Ada from E. coli, or farnasyl transferase where it has been shown that the thioether resulting from alkyl group transfer remains in the coordination sphere of the zinc. The following complexes have been structurally characterized: [(L3S)ZnI] (1), [(L3SCH(3))ZnI(2)] (2), [(L3SCH(3))ZnI]BF(4) (3), [(L3SCH(3))Zn-mu-bis-acetato-mu-hydroxo-Zn(L3SCH(3))]BF(4) (5), [(L3SCH(3))ZnSPh(F5)]ClO(4) (7), and [(L3SCH(3))(2)Zn](BF(4))(2) (8). Complexes 3, 4, 5, 7, and 8 all display thioether coordination. Thus in the absence of superior anionic ligands, thioethers are reasonably good donors to zinc in either a tetrahedral or octahedral geometry. The methylation of the complex [(L3S)ZnSPh(F5)], which contains two different thiols, produces a single product, 7, where only the aliphatic thiol has been alkylated. This observation validates the suggestion that reactivity in enzymes with multiple zinc-bound thiols could be controlled by differences in thiol pK(a) (Hammes, B. S.; Warthen, C. R.; Crans, D.; Carrano, C. J. J. Biol. Inorg. Chem. 2000, 6, 82. Compound 7 is also of interest in that it resembles the metal ion-binding site of the blue copper protein, azurin.

Alkyl and Aryl Transferases↗
PubMed2005

Ab initio based calculations of electron-transfer rates in metalloproteins.

A long-standing challenge in electron-transfer theory is to compute accurate rates of long-distance reactions in proteins. We describe an ab initio Hartree-Fock approach to compute electronic-coupling interactions and electron-transfer rates in proteins that allows the favorable comparison with experiment. The method includes the following key features; each is essential for reliable rate computations: (1) summing contributions over multiple tunneling pathways, (2) averaging couplings over thermally accessible protein conformations, (3) describing donor and acceptor electronic structure explicitly, including solvation effects, and averaging coupling over multiple energy-level crossings of the nearly degenerate donor-acceptor ligand-field states, and (4) eliminating basis set artifacts associated with diffuse basis functions. The strong dependence of coupling on donor-acceptor distance and on pathway interferences causes large variations of the computed electron-coupling values with protein geometry, and the strongest coupled conformers dominate the electron-transfer rate. As such, averaging over thermally accessible conformers of the protein and of the redox cofactors is essential. This approach was tested on six ruthenium-modified azurin derivatives using the high temperature nonadiabatic rate expression and compared with simpler pathways, average barrier, and semiempirical INDO models. Results of ab initio Hartree-Fock calculations with a split-valence basis set are in good agreement with the experimental rates. Predicted rates in the longer-distance derivatives are underestimated by 3-8-fold. This analysis indicates that the key ingredients needed for quantitatively reliable protein electron-transfer rate calculations are accessible.

Computer Simulation↗
PubMed2005

Interference, fluctuation, and alternation of electron tunneling in protein media. 1. Two tunneling routes in photosynthetic reaction center alternate due to thermal fluctuation of protein conformation.

Electron tunneling routes for the electron transfer from the bacteriopheophytin anion to the primary quinone in the bacterial photosynthetic reaction center of Rhodobactor sphaeroides are investigated by a combined method of molecular dynamics simulations for the protein conformation fluctuation and quantum chemical calculations for the electronic states of the donor, acceptor, and protein medium. The analysis of the tunneling route is made by mapping interatomic electron tunneling currents for each protein conformation. We found that there are two dominant routes mainly passing through Trp(M252) (Trp route) or mainly passing through Met(M218) (Met route). Actual electron tunneling pathways alternate between the two routes, depending on the protein conformation which varies with time. When either the Trp route or the Met route dominates, the electron tunneling matrix element /T(DA)/ becomes large. When both the Trp route and the Met route dominate, /T(DA)/ becomes very small due to the destructive interference of the electron tunneling currents between the two routes. We found that a linear relationship exists between the value of /T(DA)/ and the inverse of the degree of destructive interference Q for a wide range of values (ca. 3-10(3) for Q). A similar relationship was also found previously for electron transfer in ruthenium-modified azurins, suggesting that this relationship holds true in general. From these results, we are led to the conclusion that /T(DA)/ cannot exceed a maximum value at Q = 1, even if much variation of /T(DA)/ happens due to the fluctuation of protein conformation. We also conclude that the property of the electron transfer alternates between constructive and destructive interference, due to the fluctuation of protein conformation. It is impossible to keep a system in either constructive or destructive interference because thermal fluctuation of protein conformation takes place.

Chemistry, Physical↗
PubMed2005

Biocidal activity of a light-absorbing fluorescent conjugated polyelectrolyte.

Herein we describe studies that indicate a cationic conjugated polyelectrolyte shows biocidal activity against gram-negative bacteria (Escherichia coli, E. coli, BL21, with plasmids for Azurin and ampicillin resistance) and gram-positive bacterial spores (Bacillus anthracis, Sterne, B. anthracis, Sterne). These studies were carried out with aqueous suspensions of the conjugated polyelectrolyte, with the polyelectrolyte in supported formats and with samples in which the conjugated polyelectrolyte was coated on the bacteria. The results are interesting in that the biocidal activity is light-induced and appears effective due to the ability of the conjugated polyelectrolyte to form a surface coating on both types of bacteria. The effects observed here should be general and suggest that a range of conjugated polyelectrolytes in different formulations may provide a useful new class of biocides for both dark and light-activated applications.

Absorption↗
PubMed2003

Optimizing resolution in multidimensional NMR by three-way decomposition.

Resolution depends on the number of points sampled in a FID; in indirectly detected dimensions it is an important determinant of the total experiment time. Based on the high redundancy present in NMR data, we propose the following timesaving scheme for three-dimensional spectra. An extensive grid of discrete t1- and t2-values is used, which increases resolution while preserving the spectral width. Total experiment time is reduced by avoiding the recording of t3-FIDs for selected pairs of t1 and t2; typically the recording is omitted for about 75% of the (t1,t2) combinations. These data sets are referred to as sparse, and post-experimental processing making optimal use of spectral redundancy provides the missing, non-recorded data. We have previously shown that three-way decomposition (TWD) within the MUNIN approach provides a practical way to process dense NMR data sets. Here, a novel TWD algorithm [Ibraghimov, (2002) Numer. Linear Algebra Appl. 9, 551-565] is used to complement a sparsely recorded time-domain data set by providing the missing FIDs for all (t1,t2) combinations omitted in the experiment. A necessary condition is that for each t1-value at least a few FIDs are recorded, and similar for each t2-value. The method is demonstrated on non-uniformly sampled (15)N-NOESY-HSQC data sets recorded for the 14 kD protein azurin. The spectra obtained by TWD, reconstruction and ordinary transform to frequency-domain are, in spite of the large number of signals and the high dynamic range typical for NOESYs, highly similar to a corresponding reference spectrum, for which all (t1,t2) combinations were recorded.

Magnetic Resonance Spectroscopy↗
PubMed2006

Removal of a time barrier for high-resolution multidimensional NMR spectroscopy.

We introduce the recursive multidimensional decomposition (R-MDD) method to speed recording of high-resolution NMR spectra. The measurement time is logarithmically dependent on the sizes of indirect spectral dimensions. R-MDD has the sensitivity and resolution advantages of optimized nonuniform acquisition schemes and is applicable to all types of biomolecular spectra. We demonstrated it for triple resonance experiments on three globular proteins (ubiquitin, azurin and the barstar-barnase complex) of 8-22 kDa.

Algorithms↗
PubMed1998

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction.

Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.

Animals↗