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D Neuhaus

Publications and source records attributed to D Neuhaus.

At least 37 records · Page 2Linked to original sources
PubMed1994

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.

BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

Amino Acid Sequence↗
PubMed1994

Structure of a soluble, glycosylated form of the human complement regulatory protein CD59.

BACKGROUND: CD59 is a cell-surface glycoprotein that protects host cells from complement-mediated lysis by binding to and preventing the normal functioning of the complement proteins C8 and/or C9 which form part of a membrane penetrating assembly called the membrane attack complex. CD59 has no structural similarity to other complement proteins, but is an example of a plasma protein domain type found also in murine Ly-6 proteins and the urokinase-type plasminogen activator receptor. RESULTS: CD59 was purified from human urine, retaining the N-glycan and at least some of the non-lipid component of the glycosylphosphatidylinositol membrane anchor. The three-dimensional structure of the protein component has been determined in the presence of the carbohydrate groups using two-dimensional NMR spectroscopy. The protein structure is well defined by the NMR data (root mean square deviation from the mean structure of 0.65 A for backbone atoms and no distance constraint violations greater than 0.4 A). Structure calculations were also carried out to model the orientation of the N-acetylglucosamine residue that is directly linked to Asn18. CONCLUSIONS: The main features of the protein structure are two antiparallel beta-sheets (a central one with three strands and another with two), a short helix that packs against the three-stranded beta-sheet, and a carboxy-terminal region that, although lacking regular secondary structure, is well defined and packs against the three-stranded beta-sheet, on the opposite face to the helix. We have used the structure, in combination with existing biochemical data, to identify residues that may be involved in C8 binding.

Amino Acid Sequence↗
PubMed1994

1H NMR analysis of the partly-folded non-native two-disulphide intermediates (30-51,5-14) and (30-51,5-38) in the folding pathway of bovine pancreatic trypsin inhibitor.

The conformational properties of analogues of the (30-51,5-14) and (30-51,5-38) disulphide intermediates in refolding of reduced BPTI, with non-native second disulphide bonds, have been characterized in detail by 1H NMR analysis. They are shown to have partly-folded conformations, very similar to that of the (30-51) one-disulphide intermediate from which they arise during folding. The non-native disulphide bonds are formed in flexible or unfolded parts of the polypeptide chain; they do not disrupt the folded portion nor do they introduce substantial non-native conformation. The conformational properties of these intermediates explain their important roles in the folding pathway.

Amino Acid Sequence↗
PubMed1994

Solution structure of the DNA binding octapeptide repeat of the K10 gene product.

A putative transcription factor, the Drosophila K10 gene product, contains eight repeats of the octapeptide sequence SPNQQQHP or close variants. The solution structure of the K10 repeat was studied by NMR using a peptide composed of two SPNQQQHP units (referred to here as HP2). To overcome problems caused by degeneracy of backbone amide signals of Gln residues, a series of synthetic peptides containing an 15N-labelled main chain amide at different positions in HP2 were synthesized. In aqueous trifluoroethanol solution, HP2 folds into two structural units; the SPNQ part of each unit folds into a turn structure, while the C-terminal part shows some helical characteristics but is less structured. The N-terminal turn is likely to provide a core that produces a more stable helical structure upon binding to DNA and probably 'caps' the segmented helical unit at its N-terminus. This model is supported by a DNA footprinting study which shows that one SPNQQQHP unit spans four base pairs upon binding to A/T-rich sequences of DNA.

Amino Acid Sequence↗
PubMed1994

Haemorrheology and long term exercise.

In general, the small number of haemorrheological studies concerning acute changes in long term exercise reveals remarkable small effects, given the substantial changes of pertinent cardiorespiratory parameters. To a large extent, this appears to result from careful maintenance of extra- and intravascular water balance and adequate control of electrolytes. Haemorrheological alterations during long term exercise depend on haematological parameters, especially haematocrit and plasma protein levels, which are acutely changed. During exercise, only small deviations of haematocrit from resting values are seen in the short term. This is explained by the usually constant plasma volume which depends on the amount of fluid intake during exercise. In contrast, the exercise-induced elevation of total intravascular protein content significantly increases the levels of plasma proteins, with the exception of fibrinogen. Although this leads to an increase of plasma viscosity, the absence of substantial haematocrit changes accounts for the remarkably small alterations, if any, of blood viscosity which are observed during long term exercise. Endurance training causes haemodilution by expansion of plasma volume, thus resulting in a reduction of blood and plasma viscosity. Red cell deformability is variable with training as well as during exercise. This appears to be related to methodological problems of measurement as well as to the type of endurance exercise performed. Physiological considerations suggest that the importance, for aerobic work capacity, of haemorrheological changes occurring in long term (submaximal) exercise may be limited. By contrast, this may be different during maximal exercise activity, when the entire cardiovascular reserve has been fully recruited.

Blood Viscosity↗
PubMed1993

DNA recognition by the oestrogen receptor: from solution to the crystal.

BACKGROUND: The steroid/nuclear hormone receptors are a large family of conserved ligand-activated transcription factors that regulate gene expression through binding to response elements upstream of their target genes. Most members of this family bind to DNA as homodimers or heterodimers and recognize the sequence, spacing and orientation of the two half-sites of their response elements. The recognition and discrimination of the sequence and arrangements of these half-sites are mediated primarily by a highly conserved DNA-binding domain. RESULTS: Here we describe the DNA-binding properties of the isolated DNA-binding domain of the oestrogen receptor, the ERDBD, and its refined NMR structure. This domain is monomeric in solution, but two molecules bind cooperatively to specific DNA sequences; this cooperativity determines the arrangement of half-sites that is recognized by the ERDBD. The 10 carboxy-terminal residues and a region of 15 residues within the domain are disordered in the solution structure, yet are important for DNA binding. CONCLUSION: The cooperative nature of ERDBD binding to DNA is important. The previously-determined X-ray structure of the ERDBD dimer bound to DNA shows that the 15 internal residues disordered in solution make contact both with DNA and with the corresponding region of the other monomer. These results suggest that these residues become ordered during the process of binding to DNA, forming the dimer interface and thus contributing to the cooperative interaction between monomers.

Amino Acid Sequence↗
PubMed1993

Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. 1H and 15N resonance assignments and determination of backbone dynamics from 15N relaxation measurements.

An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by 1H and 15N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (1H)-15N NOE and 15N T1 and T2 relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The 15N relaxation data show that the N-terminal 15 residues are very flexible, and the (1H, 1H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.

Animals↗
PubMed1993

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.

Antigens, CD↗
PubMed1992

Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains.

Peptides containing either one, two or three of the three zinc-finger motifs from the yeast transcription factor SWI5 have been prepared by expression in Escherichia coli. The DNA binding characteristics of these peptides were investigated, and a two-dimensional nuclear magnetic resonance (n.m.r.) study undertaken to establish the three-dimensional structures of the two-finger peptide. The peptide containing fingers 1 and 2 binds sequence specifically to two thirds of the DNA binding site recognized either by intact SWI5 or by the isolated three-finger peptide, and hence has the correct tertiary fold for DNA recognition. These results also establish the polarity of DNA binding, since the N-terminal two fingers of SWI5 bind to the 5' end of the DNA binding site. Mild proteolysis of the three-finger peptide using trypsin results in a small number of discrete products, which is consistent with the presence of three structured mini-domains. Nearly complete n.m.r. signal assignments were obtained for two peptides containing finger 2 alone or fingers 1 + 2. Comparison of two-dimensional spectra of these peptides and others clearly shows that the NOE enhancements and chemical shifts characteristic of each finger are quite insensitive to the presence or absence of neighbouring fingers. This clearly indicates that adjacent zinc-finger domains are structurally independent in these peptides from SWI5. However, there must be some steric limitations on the possible relative orientations of the fingers, and to establish limits for these a set of structures for the peptide containing fingers 1 + 2 was calculated using the YASAP simulated annealing protocol in conjunction with n.m.r.-based constraints. A more detailed description of the three-dimensional structures of finger 1 and finger 2, and their relationship to other previously determined structures of single zinc-fingers, is given in the accompanying paper.

Amino Acid Sequence↗
PubMed1992

Solution structures of two zinc-finger domains from SWI5 obtained using two-dimensional 1H nuclear magnetic resonance spectroscopy. A zinc-finger structure with a third strand of beta-sheet.

This paper describes the detailed three-dimensional structures of two zinc-finger domains from the yeast transcription factor SWI5, calculated using the results of the n.m.r. experiments described in the accompanying paper. The structure of finger 2 is essentially similar to those previously obtained by others for isolated, synthetic single zinc-finger domains in solution, and for the three zinc-finger peptide Zif268 in its crystalline complex with DNA. The N-terminal half of the sequence forms a two-stranded, irregular beta-sheet containing both of the metal-binding cysteine residues, while the remainder of the structure forms a helix. Approximately the first half of this helix is alpha-helical, whereas the C-terminal portion, including the two metal-binding histidine residues, is 3(10) helical. Four invariant hydrophobic residues form a core to the structure. In contrast to all previously described structures of zinc-finger domains, finger 1 has an additional strand in the beta-sheet, formed by residues N-terminal to the formal start of the finger motif. This additional strand plays a role in stabilising the folded form of finger 1, since a two-finger peptide lacking the N-terminal residues showed folded structure in finger 2 but not in finger 1.

Amino Acid Sequence↗
PubMed1992

Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.

The most productive folding pathway of reduced bovine pancreatic trypsin inhibitor (BPTI) proceeds through the disulphide intermediates (30-51), (30-51, 5-14), and (30-51, 5-38); these are important kinetic intermediates in folding, even though the latter pair contain non-native disulphide bonds. Analogues of these intermediates have been prepared by protein engineering methods and their conformational properties examined by circular dichroism and 1H-nuclear magnetic resonance. The (30-51), (30-51, 5-14) and (30-51, 5-38) analogues exhibit comparable degrees of stable structure, which cannot include those portions of the polypeptide chain involving Cys5, Cys14 and Cys38. These properties are consistent with the roles of (30-51, 5-14) and (30-51, 5-38) in the folding pathway of BPTI, which demand that they exhibit a considerable degree of conformational flexibility in part of the molecule.

Animals↗
PubMed1992

Changes in haemorheology in the racing greyhound as related to oxygen delivery.

Arterial blood samples were obtained from six greyhounds during rest, immediately before, and after a 704-m (7/16th mile) race. Measurements were made of various haematological (red cell count, haemoglobin, packed cell volume, white cell count, plasma proteins) and haemorheological variables. Blood and plasma viscosity were determined at high wall shear stresses (67-200 dynes.cm-2, 670-2000 microN.cm-2) in a 20-microns glass capillary device which was designed to take the diameter dependence of blood viscosity (Fahraeus-Lindqvist effect) into account. Compared to values at rest, substantial haemoconcentration occurred before the race, mainly due to splenic discharge of red cells. Additional haemoconcentration was found after the race. The increase of effective blood viscosity caused by elevation of packed cell volume was greater than the increase in O2 binding capacity resulting from the elevated haemoglobin concentration, suggesting that the haemoconcentration observed in the exercising greyhound does not enhance O2 delivery to skeletal muscle. The main physiological effect of red cell discharge from the contracting spleen appeared to be a consequence of the volume rather than the composition of the circulating blood.

Animals↗
PubMed1992

Haemorheology and exercise: intrinsic flow properties of blood in marathon running.

Haemorheological and haematological parameters were studied in venous blood samples taken from 8 endurance-trained athletes before and after a marathon run. Viscometry was performed in a 20 microns glass capillary and in a Couette viscometer. Apparent blood viscosity was lower in the capillary than in the rotational viscometer, but significant differences between pre- and post-run samples were not observed in either system. This is presumably due to the absence of changes of haematocrit (pre-run [mean +/- SD]: 0.453 +/- 0.016, post-run: 0.456 +/- 0.018). Although protein concentration increased significantly from 70.8 +/- 2.1 g/l (pre-run) to 76.0 +/- 3.9 g/l (post-run), the difference of plasma viscosity was not statistically significant (pre-run: 1.26 +/- 0.03 cP, post-run: 1.30 +/- 0.07 cP). In all samples, plasma viscosity showed a significant positive correlation with total protein, globulin, albumin, but not with fibrinogen concentration. Cone-plate aggregometry and a filtration technique were used to evaluate red cell aggregation and deformability, two determinants of blood rheology which also were not different between pre- and post-run samples. The absence of viscosity changes in relation to the marathon run may be due to the relative stability of blood and plasma volume, which can be deduced from haematological parameters. Alterations of plasma composition (e.g. acidosis or hyperosmolality) seem to cause only minor, if any, changes of microrheological cell properties.

Adult↗
PubMed1992

Blood viscosity in tube flow: dependence on diameter and hematocrit.

Since the original publications by Martini et al. (Dtsch. Arch. Klin. Med. 169: 212-222, 1930) and Fahraeus and Lindqvist (Am. J. Physiol. 96: 562-568, 1931), it has been known that the relative apparent viscosity of blood in tube flow depends on tube diameter. Quantitative descriptions of this effect and of the dependence of blood viscosity on hematocrit in the different diameter tubes are required for the development of hydrodynamic models of blood flow through the microcirculation. The present study provides a comprehensive data base for the description of relative apparent blood viscosity as a function of tube diameter and hematocrit. Data available from the literature are compiled, and new experimental data obtained in a capillary viscometer are presented. The combined data base comprises measurements at high shear rates (u > or = 50 s-1) in tubes with diameters ranging from 3.3 to 1,978 microns at hematocrits of up to 0.9. If corrected for differences in suspending medium viscosity and temperature, the data show remarkable agreement. Empirical fitting equations predicting relative apparent blood viscosity from tube diameter and hematocrit are presented. A pronounced change in the hematocrit dependence of relative viscosity is observed in a range of tube diameters in which viscosity is minimal. While a linear hematocrit-viscosity relationship is found in tubes of < or = 6 microns, an overproportional increase of viscosity with hematocrit prevails in tubes of > or = 9 microns. This is interpreted to reflect the hematocrit-dependent transition from single- to multifile arrangement of cells in flow.

Animals↗
PubMed1991

(14-38, 30-51) double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor: a two-dimensional 1H nuclear magnetic resonance study.

An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods. The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser. Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques. The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region. The remainder of the protein is substantially identical to native BPTI. The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI.

Amino Acid Sequence↗
PubMed1991

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor.

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.

Amino Acid Sequence↗
PubMed1991

Highly selective kappa opioid analgesics. 4. Synthesis of some conformationally restricted naphthalene derivatives with high receptor affinity and selectivity.

This paper describes the synthesis and kappa and mu opioid receptor binding affinity of some conformationally restrained derivatives of the arylacetamide group in the selective kappa opioid receptor agonist (+/-)-trans-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzo [b]thiophene-4-acetamide monohydrochloride (1,PD117302), which is an analogue of U-50, 488. The methyl-substituted derivatives (+/-)-trans-N, alpha-dimethyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzo-[b] thiophene-4-acetamide monohydrochloride (6a,b) possess significantly weaker affinity than 1 for the kappa opioid receptor (Ki = 172 and 3.7 nM, respectively). It is proposed that this is due to the conformational restriction imposed by the methyl group of 6. In order to test this proposal the acenaphthene derivative and the 4,5-dihydro-3H-naphtho [1,8-bc]thiophene derivative were prepared. The acenaphthene derivative (+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro [4.5]dec-8-yl]acenaphthenecarboxamide monohydrochloride (9) was found to have high kappa opioid receptor affinity and selectivity (kappa Ki = 0.37 +/- 0.05 nM, mu/kappa = 659, delta/kappa = 1562) and is 100 times more potent than morphine as an analgesic in the rat paw pressure test for analgesia after intravenous administration (MPE50 = 0.014 and 1.4 mg/kg, respectively). The 4,5-dihydro-3H-naphtho[1,8-bc]thiophene derivative (-)-4,5-dihydro-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro [4.5]dec-8-yl]-3H-naphthol[1,8-bc]thiophene-5-carboxamide p-toluenesulfonate (17) also has high kappa opioid receptor affinity and selectivity (kappa Ki = 4.65 nM, mu/kappa = 109).

Analgesics↗