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P Bayer

Publications and source records attributed to P Bayer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure determination of the Ras-binding domain of the Ral-specific guanine nucleotide exchange factor Rlf.

Ral-specific guanine nucleotide exchange factors RalGDS, Rgl, and Rlf have been suggested to function as intermediates between Ras and Ral pathways by being able to bind Ras proteins through their C-terminal Ras-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-1 have been shown to have the same tertiary structure. In contrast to the RBDs of Raf and RalGDS, which bind either Ras or Rap with high affinity, Rlf-RBD has a similar affinity for both GTP-binding proteins. To be able to compare these RBDs on a structural level, we have solved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the betabetaalphabetabetaalphabeta-fold of the ubiquitin superfamily and is very similar to that of RalGDS-RBD. The binding interface of Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode similar to that in the case of Rap.Raf-RBD. However, comparison of the putatively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of Rlf-, RalGDS-, and Raf-RBD.

Amino Acid Sequence↗
PubMed1998

The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation.

Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescent N-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and 15N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is alpha-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.

Amino Acid Sequence↗
PubMed1998

Structure determination of the small ubiquitin-related modifier SUMO-1.

The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the betabetaalphabetabetaalphabeta fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

Amino Acid Sequence↗
PubMed1998

Equine infectious anemia virus transactivator is a homeodomain-type protein.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.

Animals↗
PubMed1998

Structure-activity relation of NH2-terminal human parathyroid hormone fragments.

Human parathyroid hormone (hPTH) is involved in the regulation of the calcium level in blood. This hormone function is located in the NH2-terminal 34 amino acids of the 84-amino acid peptide hormone and is transduced via the adenylate cyclase and the phosphatidylinositol signaling pathways. It is well known that truncation of the two NH2-terminal amino acids of the hormone leads to complete loss of in vivo normocalcemic function. To correlate loss of calcium level regulatory activity after stepwise NH2-terminal truncation and solution structure, we studied the conformations of fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison to hPTH-(1-37) in aqueous buffer solution under near physiological conditions by circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance spectroscopy, and restrained molecular dynamics calculations. All peptides show helical structures and hydrophobic interactions between Leu-15 and Trp-23 that lead to a defined loop region from His-14 to Ser-17. A COOH-terminal helix from Met-18 to at least Leu-28 was found for all peptides. The helical structure in the NH2-terminal part of the peptides was lost in parallel with the NH2-terminal truncation and can be correlated with the loss of calcium regulatory activity.

Amino Acid Sequence↗
PubMed1997

The interaction of HIV-1 Tat(32-72) with its target RNA: a fluorescence and nuclear magnetic resonance study.

We performed intrinsic peptide fluorescence experiments to characterize the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. Kd values for wild-type BP1 and cysteine-modified BP1 were found to be in the range of 60 to 70 nM for both peptides, indicating that free sulfhydryl groups of the cysteines within the peptide are not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1SW) was used to further investigate peptide RNA interaction by fluorescence studies. Titration of BP1SW with TAR resulted in a dissociation constant (Kd = 9 nM) nearly an order of magnitude lower than that of the wild-type peptide. The change of the BP1SW fluorescence intensity on TAR binding was used to investigate the kinetics of this interaction by stopped-flow experiments. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement step (k approximately 60 s-1) in order to form the specific BP1SW-TAR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1SW and BP1SW bound to TAR shows that only resonances from amino acid residues of the core and basic sequence regions are shifted on TAR binding.

Amino Acid Sequence↗
PubMed1997

Bone resorption in multiple myeloma and in monoclonal gammopathy of undetermined significance: quantification by urinary pyridinium cross-links of collagen.

To quantify osseous breakdown in multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and benign osteoporosis, we measured urinary levels of pyridinium cross-links of collagen in 50 patients with newly diagnosed and untreated MM, 40 patients with MGUS, 40 untreated patients with osteoporotic vertebral fractures, and 64 healthy adults. Ion-paired, reverse-phase high-performance liquid chromatography (HPLC) was used to measure total urinary excretion of pyridinoline (h-PYD) and deoxypyridinoline (h-DPD). Urinary excretion of free immunoreactive deoxypyridinoline (i-DPD) was determined with an enzyme immunoassay. MM patients had significantly (P < .0001) higher levels of h-PYD, h-DPD, and i-DPD than the healthy adults, patients with MGUS, or patients with osteoporosis. The MGUS and osteoporosis groups presented with elevated (P < .05) levels of urinary pyridinium cross-links when compared with healthy controls. In 20 MM patients who subsequently received chemotherapy, the percent changes in i-DPD did not correlate with the changes in the monoclonal protein. In one of three patients experiencing a transition of initial MGUS into stage I MM, i-DPD increased above the upper limit of the normal range. In 13 patients with stable MGUS, i-DPD remained normal in repeated measurements. Based on the upper limits of the normal range, the sensitivity of urinary pyridinium cross-links in stage I and II MM was low (<50%), but it was between 78% (h-DPD) and 93% (i-DPD) in stage III MM. Specificity in patients with MGUS was between 87% (h-PYD) and 97% (h-DPD). In conclusion, determining the urinary excretion of pyridinium cross-links seems to be a promising noninvasive and thus easily repeatable method for evaluating the actual degree of osseous breakdown. Although measurement of pyridinium cross-link levels is not useful in discriminating patients with MGUS from early-stage myeloma patients, determination of i-DPD levels may contribute importantly to clinical guidance, since increased i-DPD levels seem to identify patients who are particularly likely to benefit from osteoclast-inhibiting drugs such as bisphosphonates. The fact that in a number of patients paraprotein concentrations and i-DPD levels did not change in parallel but instead diverged strongly after chemotherapy might explain the observation that bone lesions sometimes progress even in patients who achieve complete remission.

Adult↗
PubMed1997

Spectroscopic investigations of HIV-1 trans-activator and related peptides in aqueous solutions.

The 86 amino acid trans-activator (Tat) protein of human immunodeficiency virus type 1 (HIV-1) is an RNA-binding transcriptional regulator. HIV-1 Tat proteins (wild type and Thr40Lys mutant) and the HIV-1 Tat peptide fragments Tat(32-48) and Tat(32-72) were chemically synthesized. One- and two-dimensional nuclear magnetic resonance spectroscopy experiments were performed to elucidate the structural features of these proteins. In fluorescence quenching studies of the full-length Tat protein (Thr40Lys), Trp11 was found to be only partially protected against solvent accessibility. Circular dichroism melting studies monitored a slight cooperative change in the conformation of the Tat with increasing temperature. Backbone NH protons of amino acids located in the main core element of the protein are partially protected against exchange.

Amino Acid Sequence↗
PubMed1997

Whole-blood immunoassay (SimpliRED) versus plasma immunoassay (NycoCard) for the diagnosis of clinically suspected deep vein thrombosis.

BACKGROUND: The level of D-dimer in the blood reflects the level of lysed, cross-linked fibrin, and is a useful diagnostic marker in patients with clinically suspected deep vein thrombosis (DVT). In this study, two assays for the measurement of D-dimer levels were compared: the new, whole-blood immunoassay, SimpliRED, which can be performed at the patient's bedside in two minutes; and the plasma immunoassay, NycoCard. PATIENTS AND METHODS: D-dimer levels were determined using these two techniques in 108 patients with clinically suspected DVT. To ascertain or rule out the diagnosis of DVT compression ultrasonography using a colour coded Duplex instrument was done. 8 doubtful cases were diagnosed by ascending phlebography. By these procedures DVT was confirmed in 33 patients and ruled out in the remaining 75 cases. RESULTS: The SimpliRED assay exhibited a sensitivity of 1.0 (CI 0.89-1.0) and a specificity of 0.75 (CI 0.63-0.84); negative predictive value 1.0 (CI 0.94-1.0), positive predictive value 0.63 (CI 0.49-1.0). By contrast, the NycoCard assay displayed a sensitivity of 0.85 (CI 0.68-0.95) and a specificity of 0.65 (CI 0.53-0.76); negative predictive value 0.91 (CI 0.80-0.97), positive predictive value of 0.52 (CI 0.38-0.66). CONCLUSION: The results show that the SimpliRED assay is a valuable tool in the diagnosis of clinically suspected DVT, especially when laboratory facilities are not accessible.

Adult↗
PubMed1997

[Inflammatory smears in cervicovaginal cytology. A finding meaning infection?].

INTRODUCTION: Inflammatory changes are relatively frequent findings in cervical smears and are generally believed to be a consequence of genital infection. However, clinical signs of infection are frequently absent and no consensus exists on the management of these patients. The objective of this study was to assess whether or not inflammatory smears are exclusively a consequence of genital infection. For this purpose, the prevalence of genital infection in a group of women with inflammatory cells in the cervical smear and a control group with normal smears was compared. MATERIAL AND METHODS: Sixty-two regularly menstruating women aged 17 to 48 years, attending the outpatient Gynaecology sector of S. João Hospital, were prospectively evaluated. Cervical smears were analysed by the same cytologist who chose 10 inflammatory cells per high power field (400x) as the cut-off value for normality. Infection by Candida spp., Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasma hominis, Ureaplasma urealyticum, Chlamydia trachomatis and bacterial vaginosis was investigated. Human Papillomavirus infection was evaluated by colposcopy and biopsy of abnormal colposcopic findings. RESULTS: Thirty-four women and inflammatory cells on their cervical smear and 15 of these (44%) had a genital infection. Of the 28 women with normal smears, 12 (43%) had a genital infection. No statistically significant difference in the prevalence of infection was found between the two groups. CONCLUSIONS: Our results suggest that the presence of inflammatory cells in cervical smears is not necessarily due to infection and other causes may be responsible for their appearance.

Adolescent↗
PubMed1996

Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.

Amino Acid Sequence↗
PubMed1996

Secondary structure of an armadillo single repeat from the APC protein.

The armadillo domain is a repeating sequence motif of a variety of proteins with different functions. Here we describe the structure of a synthetic single armadillo repeat solved by two-dimensional nuclear magnetic resonance spectroscopy. Our results indicate alpha-helical secondary structural elements in half of the residues.

Adenomatous Polyposis Coli Protein↗
PubMed1995

Structure of amyloid A4-(1-40)-peptide of Alzheimer's disease.

One of the principle peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40-amino-acid peptide beta-amyloid A4-(1-40)-peptide. The full-length A4-(1-40)-peptide was chemically synthesized and the solution structure determined by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular-dynamics calculations. Synthetic human A4-(1-40)-peptide was soluble and non-aggregating for several days in 40% (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium-range cross-peaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long-range NOE cross-peaks that would indicate tertiary structure of the peptide were absent. The main secondary-structure elements found by chemical-shift analysis, sequential and medium-range NOESY data, and NOE-based restrained molecular-dynamics calculations were two helices, Gln15-Asp23 and Ile31-Met35, whereas the rest of the peptide was in random-coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4-(1-40)-peptide.

Alzheimer Disease↗
PubMed1995

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Amino Acid Sequence↗
PubMed1995

Structural studies of HIV-1 Tat protein.

Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). Tat proteins are thus ideal targets for drugs intervening with lentiviral growth. The consensus RNA binding motif (TAR, trans-activation responsive element) of HIV-1 is well characterized. Structural features of the 86 amino acid HIV-1, Zaire 2 isolate (HV1Z2) Tat protein in solution were determined by two dimensional (2D) nuclear magnetic resonance (NMR) methods and molecular dynamics (MD) calculations. In general, sequence regions corresponded to structural domains of the protein. It exhibited a hydrophobic core of 16 amino acids and a glutamine-rich domain of 17 amino acids. Part of the NH2 terminus, Val4 to Pro14, was sandwiched between these domains. Two highly flexible domains corresponded to a cysteine-rich and a basic sequence region. The 16 amino acid sequence of the core region is strictly conserved among the known Tat proteins, and the three-dimensional fold of these amino acids of HV1Z2 Tat protein was highly similar to the structure of the corresponding EIAV Tat domain. HV1Z2 Tat protein contained a well defined COOH-terminal Arg-Gly-Asp (RGD) loop similar to the recently determined decorsin RGD loop.

Amino Acid Sequence↗
PubMed1995

Nonenzymatic hydrolysis of adenosinetriphosphate (ATP) at high temperatures and high pressures.

Marine organisms living in the deep sea near to hot wells show a fascinating tolerance to extremely high temperatures and pressures. Under the given conditions the synthesis and stability of biomolecules seem to be limiting facts for the basis of life. We studied the influence of high pressures and high temperatures on the hydrolysis of ATP, a universal component for the storage of energy in all known organisms and therefore an extremely interesting and important molecule. The hydrolysis of ATP, ADP and AMP was studied in unbuffered solutions at temperatures between 353 and 369 K at pH values between 3.4 and 10.0. The pressure dependence was determined to p(max) = 220 MPa also at pH 5. All data can be explained by a proton catalyzed mechanism that removes in consecutive steps the final phosphate group. In none of the experiments could pyrophosphate be detected. The influence of phosphate and magnesium ions on the hydrolysis is discussed.

Journal Article↗