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H Fabian

Publications and source records attributed to H Fabian.

At least 37 records · Page 2Linked to original sources

Aspartate-bond isomerization affects the major conformations of synthetic peptides.

The aspartic acid bond changes to an beta-aspartate bond frequently as a side-reaction during peptide synthesis and often as a post-translational modification of proteins. The formation of beta-asparate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic-acid-bond isomerization may help the selective aggregation and retention of the amyloid beta peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fourier-transform infrared spectroscopy, and molecular modeling to characterize the general effect of the beta-aspartate-bond formation on the conformation of five sets of synthetic model peptides. Each of the non-modified, parent peptides has one of the major secondary structures as the dominant spectroscopically determined conformation: a type I beta turn, a type II beta turn, short segments of alpha or 3(10) helices, or extended beta strands. We found that both types of turn structures are stabilized by the aspartic acid-bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid-chain broke both the helix and the beta-pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid-bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post-translational modifications in defining the secondary structure of protein fragments.

Amino Acid Sequence↗

Isotope-edited Fourier transform infrared spectroscopy studies of calmodulin's interaction with its target peptides.

The ubiquitous calcium-binding protein calmodulin (CaM) regulates a wide variety of cellular events by binding to and activating many distinct target enzymes. The CaM-binding domains of most of these enzymes are contained in a contiguous stretch of amino acids with a length of approximately 20 residues. In this work, we have used "isotope-edited" Fourier transform infrared spectroscopy to study the interaction of CaM with synthetic peptides resembling the CaM-binding domains of myosin light chain kinase (MLCK), constitutive nitric oxide synthase (cNOS), and caldesmon (CaD). Uniform labeling of CaM with carbon-13 causes the amide I band of the protein to shift approximately 55 cm-1 to lower frequency in D2O, leaving a clear window in the infrared spectrum for observing the amide I band of the unlabeled target peptides. Upon complex formation, the amide I bands of the CaM-binding domains of MLCK and cNOS shift 4 cm-1 toward higher frequency (to approximately 1648 cm-1), and have a narrower bandwidth compared to the peptide in aqueous solution. These spectral changes and the fact that the infrared spectra of these two peptides in their complex with CaM closely resemble those recorded in a mixture of D2O and the helix inducing solvent trifluoroethanol indicate that they bind to CaM in an alpha-helical conformation. The CaM-binding domain of CaD also showed similar, but less dramatic, spectral changes; this is in agreement with the fact that it binds to CaM with lower affinity and a shorter alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by Fourier transform infrared spectroscopy.

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus oryzae↗

Zinc chelation and structural stability of adenylate kinase from Bacillus subtilis.

Adenylate kinase from Bacillus subtilis, like the enzyme from Bacillus stearothermophilus, contains a structural zinc atom. Cys153 in the enzyme from B. stearothermophilus, which is involved in the zinc coordination, is replaced in the adenylate kinase from B. subtilis by an aspartic acid residue. Therefore, we were interested in establishing whether this difference has an impact on the structure, the metal chelation, and the overall stability of these proteins. We also were interested in determining whether His138, which is conserved in many adenylate kinases, can act as a fourth partner in the metal chelation and, in general, whether His can successfully replace Cys or Asp in coordinating zinc in the adenylate kinase from B. subtilis. The adk gene from B. subtilis was cloned by polymerase chain reaction. The wild-type protein, together with several variants obtained by site-directed mutagenesis, were expressed in Escherichia coli and analyzed by biochemical and physicochemical methods. The H138N and D153C mutants of adenylate kinase from B. subtilis exhibited properties similar to those of the wild-type protein, indicating that His138 is not involved in metal coordination and that Asp153, just like Cys in the analogous position in the enzyme from B. stearothermophilus, can participate in zinc chelation. This is the first experimental evidence indicating that aspartic acid can be involved in the coordination of a structural zinc atom. On the other hand, the D153H and D153T variants showed significant changes in their zinc-binding properties. Dialysis of the latter proteins against buffer (in both the presence and the absence of 2 mM EDTA) resulted in removal of the metal ion and loss of enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Kinase↗

Synthetic post-translationally modified human A beta peptide exhibits a markedly increased tendency to form beta-pleated sheets in vitro.

The beta-amyloid peptide (A beta) is the major constituent of senile plaques, one of the hallmark neuropathological lesions of Alzheimer's disease. Recently a post-translationally modified analogue of the human beta-amyloid peptide, which contains isoaspartic residues in positions 1 and 7, was isolated from parenchyma and leptomeningeal microvasculature of Alzheimer's disease patients [Roher, A. E., Lowenson, JD., Clarke, S., Wolkow, C., Wang, R., Cotter, R. J., Reardon, I. M., Zürcher-Neely, H. A., Heinrikson, R. L., Ball, M. J. & Greenberg, B. D. (1993) J. Biol. Chem. 268, 3072-3083]. We used circular dichroism and Fourier-transform infrared spectroscopy to characterize the conformational changes on human A beta upon substitution of Asp1 and Asp7 to isoaspartic residues. We found that the intermolecular beta-pleated-sheet content is markedly increased for the post-translationally modified peptide compared to that in the corresponding unmodified human or rodent A beta sequences both in aqueous solutions in the pH 7-12 range, and in membrane-mimicking solvents (such as aqueous octyl-beta-D-glucoside or aqueous acetonitrile solutions). These findings underline the importance of the originally alpha-helical N-terminal regions of the unmodified A beta peptides in defining its secondary structure and may offer an explanation for the selective aggregation and retention of the isomerized A beta peptide in Alzheimer's-disease-affected brains.

Amino Acid Sequence↗

Tyrosine- versus serine-phosphorylation leads to conformational changes in a synthetic tau peptide.

One of the major immunodominant epitopes of the paired helical filaments (PHF) of Alzheimer's disease is the peptide sequence GAEIVYKSPVVSGD (T3), comprising amino acids 389-402 of the microtubule-associated protein, tau, when it is phosphorylated at the first serine residue. While the corresponding anti-PHF monoclonal antibody recognizes the peptide phosphorylated at either serine, it does not recognize the tyrosine-phosphorylated peptide. Here we describe the effect of serine- versus tyrosine-phosphorylation on the conformation of a synthetic tau peptide. While adding a phosphate to the serine residue has practically no impact on the structure of the non-phosphorylated peptide, phosphorylation of the tyrosine results in considerable conformational changes.

Alzheimer Disease↗

Structural and physico-chemical characteristics of Bordetella pertussis adenylate kinase, a tryptophan-containing enzyme.

The adk gene from the Gram-negative pathogen Bordetella pertussis was cloned by complementing the thermosensitive Escherichia coli adk strain CR341T28. B. pertussis adenylate kinase is a 218-amino-acid protein that has high similarity with adenylate kinase from Escherichia coli and Hemophilus influenzae (57%). A distinct characteristic of enzyme from B. pertussis, not found in other bacterial adenylate kinases, is the presence of a tryptophan residue at position 185. Although distant from the catalytic site, this single tryptophan serves as a convenient probe for monitoring the binding of nucleotide substrates or analogs to the enzyme. Differential scanning calorimetry and equilibrium unfolding experiments in guanidine.HCl indicate similar stabilities for adenylate kinase from B. pertussis and E. coli. An extensive comparison between physico-chemical properties of adenylate kinase from B. pertussis and the enzyme from E. coli showed that the kinetic and structural properties of the two enzymes are very similar. However, infrared spectroscopy has allowed to identify small but significant differences in the secondary structure of the two proteins.

Adenylate Kinase↗

Secondary structure and temperature-induced unfolding and refolding of ribonuclease T1 in aqueous solution. A Fourier transform infrared spectroscopic study.

The secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% alpha-helix and 32 to 33% beta-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with the crystal structure which indicates 16% alpha-helix and 35% beta-sheet. Specific IR-marker bands are assigned to the different beta-sheet structures, to the slightly bent alpha-helix, and to beta-turn and irregular conformations present in RNase T1. The temperature dependence of the infrared spectra shows that the thermal unfolding and refolding of RNase T1 is fully reversible. This permitted the detailed analysis of structural changes that occur as a function of temperature by evaluating quantitatively the various secondary structure-related amide I band components and some amino acid side-chain vibrations as specific monitors. The secondary structure of RNase T1 is essentially retained in the temperature range between 20 and 50 degrees C. Significant perturbation of protein structure is initiated between 50 and 55 degrees C within regions of beta-sheet structures while the alpha-helix remains virtually intact up to 55 degrees C suggesting a "premelting" of RNase T1. Between 55 and 60 degrees C, a highly co-operative unfolding process is indicated by the simultaneous breakdown of all secondary structure components and by distinct changes of some specific side-chain vibrations. An analysis of the amide I band contour of RNase T1 at 70 degrees C proves that the unfolded state is predominantly, but not completely, irregular or "random coil". Residual, turn-like structures persisting even in the unfolded state are suggested by minor, turn related band components in the amide I region. From IR-spectra collected along a linear temperature gradient, intensity/temperature and frequency/temperature profiles were constructed using some peptide backbone and amino acid side-chain marker bands as local, structure-sensitive monitors. From these profiles individual transition temperatures tm and transition enthalpies delta H (van't Hoff) were calculated. The tm and delta H values revealed a small but distinct hysteresis between repetitive cycles of unfolding and refolding of the protein, suggesting slow refolding kinetics of RNase T1. Furthermore, the various infrared "marker bands" indicate a slightly different response towards temperature increase/decrease for different regions of the protein. The data demonstrate that infrared spectroscopy permits both the detailed analysis of structural changes occurring in a protein as a function of temperature and the determination of thermodynamic parameters characterizing its folded/unfolded state transition.

Amides↗

Comparative analysis of human and Dutch-type Alzheimer beta-amyloid peptides by infrared spectroscopy and circular dichroism.

The 42 amino acid beta A4 peptide is the major constituent of the senile plaques, one of the hallmark neuropathological lesions of Alzheimer's disease. While C-terminally truncated variants were shown to be present in normal body fluids, a single Glu-->Gln change in the 39 amino acid form of beta A4 results in accelerated fibril formation in the brains of patients with Dutch-type hereditary cerebral hemorrhage with amyloidosis. In this study we used Fourier-transform infrared and circular dichroism spectroscopies on synthetic peptides to demonstrate that this mutation results in altered secondary structure in membrane mimicking solvents, characterized by a considerably higher beta-structure content for the mutant peptide. Moreover, extreme high and low pH were less effective in eliminating the beta-conformation for the Dutch-variant than for the normal human sequence.

Alzheimer Disease↗

Conformation of d(GGGATCCC)2 in crystals and in solution studied by X-ray diffraction, Raman spectroscopy and molecular modelling.

In the crystal, d(GGGATCCC)2 forms an A-DNA double helix as known from a single crystal X-ray diffraction study. Accordingly, in the Raman spectra of crystals the A-family marker bands at 664, 705, 807 and 1101 cm-1 and the spectral characteristics in the region 1200 to 1500 cm-1 clearly demonstrate the A-form as the dominant conformation. Bands at 691, 850, and 1080 cm-1, however, indicate that a minor fraction of the octamer molecules in the crystal is in an unusual, still not unequivocally identified conformation possibly belonging to the B-family. In solution, the octamer is in B-like conformation as shown by the presence of B-DNA Raman marker bands at 685, 837, 1094 and 1421 cm-1. Molecular modelling techniques lead to three structures with slightly different B-form geometries as the lowest energies models when a sigmoidal dielectric function with the bulk dielectric constant epsilon = 78 and the value q = -0.5e for the effective phosphate charges was used in the calculations. An A-form structure bearing a strong resemblance to the experimentally determined crystal structure becomes the lowest energy model structure when the electrostatic parameters are changed to epsilon = 30 and q = -0.25e, respectively.

Base Sequence↗

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.

Amides↗

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.

Amino Acid Sequence↗

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.

Amino Acids↗

Pharmacokinetics of iloprost in patients with chronic renal failure and on maintenance haemodialysis.

Iloprost is a potent, chemically stable prostacyclin-mimetic for which therapeutic efficacy has been proven in patients with peripheral arterial occlusive disease (PAOD) and in those suffering from Raynaud's phenomenon. In volunteers and PAOD-patients the pharmacokinetics of iloprost after intravenous (i.v.) infusion treatment was characterized by dose-dependent steady-state plasma levels, a terminal half-life of approximately 20-30 min, and a total clearance of 15-20 ml/min/kg. Bioinactivation was mainly due to beta-oxidation. In the present study the pharmacokinetics of iloprost was investigated in 21 patients suffering from renal insufficiency, which either required haemodialysis or not. They were treated by one hour i.v. infusion with 1 ng/kg/min and blood samples were taken during and after the end of infusion. Due to technical sampling problems iloprost pharmacokinetics could only be calculated for seven dialysis and eight non-dialysis patients. In the dialysis patients steady-state levels were 114 to 320 pg/ml as compared to 36 to 70 pg/ml in the non-dialysis group. Half-lives were similar in both groups: alpha-phase: 0.05 h and beta-phase: 0.5 h. The total clearance was 2.6 to 8.0 ml/min/kg (dialysis patients) and 13.2 to 25.8 ml/min/kg (non-dialysis patients). The present study demonstrated that the pharmacokinetic profile of iloprost in patients with renal failure (not subject to haemodialysis) was similar to that observed in PAOD-patients and volunteers. In patients on maintenance haemodialysis, iloprost clearance was reduced by a factor of four. The iloprost dose regimen required in general (due to interindividual variability in response) a careful dose titration.

Adult↗

Infrared data indicate U.U mispairs in rat liver 5S RNA.

The infrared spectra of ribosomal 5S RNAs of Escherichia coli and rat liver were measured under conditions where the structure of 5S RNA is preserved. The comparison of the spectra shows significant spectral differences for the U residues which resemble in their characteristics the infrared spectra of polyuridylic acid in its base paired state. This strongly indicates the presence of U.U mispairs in rat liver 5S RNA. Juxtaposed U residues at positions U80-U96 and U73-U102 are potential candidates for U.U mispairs in rat liver 5S RNA.

Animals↗

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA.

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

Adenine↗

Laser Raman studies of the 5 S rRNA-protein L5 complex of rat liver ribosomes.

The effects of ribosomal protein L5 on the conformation of 5 S rRNA in the 5 S rRNA-protein L5 complex extracted from rat liver ribosomes have been studied by laser Raman spectroscopy. A comparison of the spectra shows small protein-induced conformational changes in the 5 S rRNA, but most of the base-paired regions appear to be present in the complex with protein L5 as well as in the free 5 S rRNA. Furthermore specific interactions between 5 S rRNA and protein L5 are indicated. Cytosine (and/or uracil) residues in single-stranded regions and the N(7) of guanine are engaged in interactions with the protein as suggested by the Raman data.

Animals↗

Universal structural features of prokaryotic and eukaryotic ribosomal 5S RNA derived from comparative analysis of their sequences.

An extensive comparative analysis of more than fifty available sequences of ribosomal 5S RNA has been made. Both for prokaryotic and eukaryotic 5S RNA a generalized secondary structure is presented which is similar to that suggested by Nishikawa and Takemura modified in few positions only. Both generalized secondary structures contain five main helical regions and a high base-pairing content of about 65 +/- 5%. The general structural architecture of prokaryotic and eukaryotic 5S RNA molecules appears to be very similar with minor modifications within particular subgroups of organisms. Conserved and semiconserved nucleotides are accumulated in the single stranded parts of 5S RNA. Functional importance was suggested for some of these regions; other short conserved nucleotide stretches may be involved in the folding of 5S RNA molecules. In particular, we propose a tertiary base-pairing interaction between the universal invariant GUA sequence (positions 76-78 and 75-77 in prokaryotic and eukaryotic 5S RNA, respectively) and the complementary conserved CPuU sequence (positions 38-40 and 36-38) in a parallel manner. A molecular model of the 5S RNA of human KB cells was constructed, which verifies the proposed tertiary interaction, probably stabilizing the two neighboured helices E and D and a stacking arrangement of the bases in the sequence positions 67-108 (and 70-106) in eukaryotic (and prokaryotic) 5S RNAs, respectively.

Animals↗