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D Neuhaus

Publications and source records attributed to D Neuhaus.

At least 19 recordsLinked to original sources
PubMed2001

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase.

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

Amino Acid Sequence↗
PubMed2001

Solution studies of chymotrypsin inhibitor-2 glutamine insertion mutants show no interglutamine interactions.

Mutants of chymotrypsin inhibitor protein 2 have previously been studied in which 4 or 10 glutamine residues were inserted into the inhibitory loop of the protein between residues 59 and 60, as potential models for the behaviour of glutamine tracts in proteins associated with polyglutamine-expansion neurodegenarative diseases. These mutants form very stable monomers, dimers and trimers. Although the cause of oligomerisation was found to be domain-swapping, it was thought that the glutamine insertions might nevertheless show evidence of weak interglutamine interactions in solution that could mimic those occurring in disease-associated proteins. In the present NMR study, we used steady-state (15)N[(1)H] NOE measurements and chemical shift comparisons to characterise the motional properties of the inserted glutamines in these CI2 mutants. We found the glutamines to be highly mobile, with no evidence of interactions amongst them in either monomers or dimers.

Amino Acid Sequence↗
PubMed2001

HMG-D complexed to a bulge DNA: an NMR model.

An NMR model is presented for the structure of HMG-D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.

Animals↗
PubMed2000

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

Acetylation↗
PubMed2000

Growth and form of planetary seedlings: results from a microgravity aggregation experiment.

The outcome of the first stage of planetary formation, which is characterized by ballistic agglomeration of preplanetary dust grains due to Brownian motion in the free molecular flow regime of the solar nebula, is still somewhat speculative. We performed a microgravity experiment flown onboard the space shuttle in which we simulated, for the first time, the onset of free preplanetary dust accumulation and revealed the structures and growth rates of the first dust agglomerates in the young solar system. We find that a thermally aggregating swarm of dust particles evolves very rapidly and forms unexpected open-structured agglomerates.

Models, Theoretical↗
PubMed2000

The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein.

The status of the poly(A) tail at the 3'-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3'-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

3' Untranslated Regions↗
PubMed1998

Solution structure of the motile major sperm protein (MSP) of Ascaris suum - evidence for two manganese binding sites and the possible role of divalent cations in filament formation.

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many other amoeboid cells. MSP is a dimeric molecule that polymerizes to form non-polar filaments constructed from two helical subfilaments that wind round one another. Moreover, MSP filaments can interact with one another to form higher-order assemblies without requiring the range of accessory proteins usually employed in actin-based systems. A knowledge of how MSP polymerizes and forms the hierarchical series of helical MSP macromolecular assemblies is fundamental to understanding locomotion in these cells. Here we describe the solution structure of MSP dimers determined by NMR spectroscopy under conditions where MSP does not polymerize to form filaments. The solution structure is indistinguishable from that observed in putative MSP subfilament helices by X-ray crystallography, indicating that MSP polymerization is not accompanied by a major conformational change. We also show that the rate of MSP polymerization associated with movement of vesicles in an in vitro motility assay is enhanced by the presence of magnesium and manganese ions and use NMR to show that the primary residues that bind these ions are 24-25 and 83-86. These residues are distant from the interface formed between MSP dimers in subfilament helices, and so are probably not involved in this level of polymerization. Instead the manganese and magnesium ion binding appears to be associated with the assembly of subfilaments into filaments and their subsequent aggregation into bundles.

Animals↗
PubMed1998

Determination of the NMR structure of the complex between U1A protein and its RNA polyadenylation inhibition element.

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.

Amino Acid Sequence↗
PubMed1997

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Amino Acid Sequence↗
PubMed1996

The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution.

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.

Actin Cytoskeleton↗
PubMed1996

The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold.

BACKGROUND: The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition. The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements. RESULTS: Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures. Sequence analysis suggests that other zinc finger proteins may also have this structure. Biochemical studies show that this additional structure increases DNA-binding affinity. CONCLUSIONS: The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold. This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.

Amino Acid Sequence↗
PubMed1996

Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation.

Many proteins involved in pre-mRNA processing contain one or more copies of a 70-90-amino-acid alphabeta module called the ribonucleoprotein domain. RNA maturation depends on the specific recognition by ribonucleoproteins of RNA elements within pre-mRNAs and small nuclear RNAs. The human U1A protein binds an RNA hairpin during splicing, and regulates its own expression by binding an internal loop in the 3'-untranslated region of its pre-mRNA, preventing polyadenylation. Here we report the nuclear magnetic resonance structure of the complex between the regulatory element of the U1A 3'-untranslated region (UTR) and the U1A protein RNA-binding domain. Specific intermolecular recognition requires the interaction of the variable loops of the ribonucleoprotein domain with the well-structured helical regions of the RNA. Formation of the complex then orders the flexible RNA single-stranded loop against the protein beta-sheet surface, and reorganizes the carboxy-terminal region of the protein to maximize surface complementarity and functional group recognition.

Amino Acid Sequence↗
PubMed1996

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Amino Acid Sequence↗
PubMed1995

The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium.

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.

Animals↗
PubMed1995

Evidence for an unusual ZW/ZW'/ZZ sex-chromosome system in Scardinius erythrophthalmus (Pisces, Cyprinidae), as detected by cytogenetic and H-Y antigen analyses.

Fifty-seven individuals of the European cyprinid fish species Scardinius erythrophthalmus were sexed by gonad histology, and their karyotypes were analyzed by Giemsa staining and C-banding. The chromosome number in somatic metaphase plates was 2n = 50. Karyotypes of all gonadal male animals were identical, consisting of 48 small meta- or submetacentric chromosomes and a pair of large metacentric chromosomes (ZZ males). Of the 33 gonadal females, 16 had a karyotype similar to the males' (ZW' females) and 17 had a heteromorphic pair of chromosomes, including a large metacentric Z chromosome and a small acrocentric W chromosome (ZW females). H-Y antigen typing with cells obtained from a variety of tissues revealed that the homogametic ZZ males were H-Y negative and the heterogametic ZW females were H-Y positive in all tissues tested. In contrast, ZW' females showed no expression of the H-Y antigen in somatic cells but were H-Y positive in ovarian cells. These results suggest that the ZW/ZW'/ZZ sex chromosomes of S. erythrophthalmus represent an unusual system of sex determination and that H-Y antigen expression is coupled with the heterogametic sex. Whether the ZW' individuals are indeed heterogametic females or actually sex-reversed ZZ males, in which chromosomal sex determination is overruled by external factors, is discussed.

Animals↗
PubMed1995

Fluid, electrolyte, and packed cell volume shifts in racing greyhounds.

Arterial blood samples were obtained at rest, just before, and 5 minutes after a 704-m race, to quantify changes in hematologic variables, plasma electrolyte and protein concentrations, osmolality, and acid/base variables. Changes in plasma volume were estimated from the change in plasma protein concentration. Immediately prior to the race, plasma volume decreased by 10% from rest and total circulating RBC volume increased by 60%, attributable to increased RBC number rather than size. Increases in blood volume (VB) by 24% and PCV by 29% also were detected before the race. Five minutes after the race, plasma volume was 21% below the resting value and total circulating RBC volume had increased 73% above the resting value, resulting in a 40% increase in PCV. Contraction of the spleen appeared responsible for increased PCV and VB before the race and maintenance of VB after the race. Plasma chloride concentration was the same before and after the race; the chloride content of the plasma decreased by the same fraction (22%) as did the plasma volume, indicating Cl- loss from the plasma. Plasma Na+ content decreased by a smaller fraction (13%), causing Na+ concentration to increase from 151 mEq/L at rest to 167 mEq/L after the race. Assuming that Na+ concentration was the same throughout the extracellular fluid, H2O likely moved into the intracellular compartment. As a consequence of these changes, the inorganic strong ion difference in plasma increased by about 16 mEq/L, tending to minimize the acid/base disturbance induced by the 33 mEq/L increase in lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1994

NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.

Amino Acid Sequence↗