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C Arrowsmith

Publications and source records attributed to C Arrowsmith.

9 recordsLinked to original sources
PubMed2005

ABACUS, a direct method for protein NMR structure computation via assembly of fragments.

The ABACUS algorithm obtains the protein NMR structure from unassigned NOESY distance restraints. ABACUS works as an integrated approach that uses the complete set of available NMR experimental information in parallel and yields spin system typing, NOE spin pair identities, sequence specific resonance assignments, and protein structure, all at once. The protocol starts from unassigned molecular fragments (including single amino acid spin systems) derived from triple-resonance (1)H/(13)C/(15)N NMR experiments. Identifications of connected spin systems and NOEs precede the full sequence specific resonance assignments. The latter are obtained iteratively via Monte Carlo-Metropolis and/or probabilistic sequence selections, molecular dynamics structure computation and BACUS filtering (A. Grishaev and M. Llinás, J Biomol NMR 2004;28:1-10). ABACUS starts from scratch, without the requirement of an initial approximate structure, and improves iteratively the NOE identities in a self-consistent fashion. The procedure was run as a blind test on data recorded on mth1743, a 70-amino acid genomic protein from M. thermoautotrophicum. It converges to a structure in ca. 15 cycles of computation on a 3-GHz processor PC. The calculated structures are very similar to the ones obtained via conventional methods (1.22 A backbone RMSD). The success of ABACUS on mth1743 further validates BACUS as a NOESY identification protocol.

Algorithms↗
PubMed2001

The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding.

Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). Correlation analysis of the amide (1)H and (15)N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70(1-326) with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70(1-326), indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70(1-326) and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70(1-168)). We also examined the structure of hRPA70(1-326) after addition of three different ssDNA substrates. Each of these substrates induced specific amide (1)H and/or (15)N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide (1)H and (15)N chemical shift changes of hRPA70(1-168) after addition of ssDNA. The same amide (1)H and (15)N chemical shift changes were observed for the NTD in hRPA70(1-168) and hRPA70(1-326). The NTD residues with the largest amide (1)H and/or (15)N chemical shift changes were localized to a basic cleft that is important for hRPA70-protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70-protein interactions.

Amides↗
PubMed2001

Analysis of aggregation, a worked example: numbers of ticks on red grouse chicks.

The statistical aggregation of parasites among hosts is often described empirically by the negative binomial (Poisson-gamma) distribution. Alternatively, the Poisson-lognormal model can be used. This has the advantage that it can be fitted as a generalized linear mixed model, thereby quantifying the sources of aggregation in terms of both fixed and random effects. We give a worked example, assigning aggregation in the distribution of sheep ticks Ixodes ricinus on red grouse Lagopus lagopus scoticus chicks to temporal (year), spatial (altitude and location), brood and individual effects. Apparent aggregation among random individuals in random broods fell 8-fold when spatial and temporal effects had been accounted for.

Animals↗
PubMed2000

Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression.

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

Adenoviridae↗
PubMed1994

A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes.

A pulsed field gradient three-dimensional isotope-filtered 13C HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp-repressor, its natural abundance co-repressor, L-tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra- and intermolecular NOEs in the isotope-edited 3D 13C HMQC-NOESY spectrum of the complex.

Bacterial Proteins↗
PubMed1994

The solution structures of the trp repressor-operator DNA complex.

The solution structures of the complex between Escherichia coli trp holorepressor and a 20 base-pair consensus operator DNA were determined. The majority of proton chemical shifts of the trp holorepressor and operator DNA were assigned from homonuclear 2D NOESY spectra of selectively deuterated analog-operator DNA complexes and the 3D NOESY-HMQC spectrum of a uniformly 15N-labeled repressor-operator DNA complex. The structures were calculated using restrained molecular dynamics and sequential simulated annealing with 4086 NOE and other experimental constraints. The root-mean-squared deviation (RMSD) among the calculated structures and their mean is 0.9(+/- 0.3)A for the repressor backbone, 1.1(+/- 0.5)A for the DNA backbone, and 1.3(+/- 0.3)A for all heavy atoms. The DNA is deformed to a significant extent from the standard B DNA structure to fit the helix-turn-helix (HTH) segment of the repressor (helices D and E) into its major grooves. Little change is found in the ABCF core of the repressor on complexation in comparison to the free repressor, but changes in the cofactor L-tryptophan binding pocket and the HTH segment are observed. The N-terminal residues (2 to 17) are found to be disordered and do not form stable interactions with DNA. Direct H-bonding to the bases of the operator DNA is consistent with all of our observed NOE constraints. Hydrogen bonds from NH eta 1 and NH eta 2 of Arg69 to O-6 and N-7 of G2 are compatible with the solution structure, as they are with the crystal structure. Other direct H-bonds from Lys72, Ala80, Ile79, Thr83 and Arg84 to base-pair functional groups can also be formed in our solution structures.

Amino Acid Sequence↗
PubMed1991

The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution.

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10. Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Segmental differences in the stability of the trp-repressor peptide backbone.

Exchange lifetimes of amide protons in trp-repressor with and without the corepressor, L-tryptophan, were studied by heteronuclear 2D NMR spectroscopy. The amide proton exchange times revealed pronounced differences in the stability of different regions of the trp-repressor. The dimeric core of the molecule is relatively compact and homogeneous in terms of the measured parameters in both apo- and holorepressors. On the other hand the DNA-binding region appears less stable and more susceptible to the exchange of its backbone protons with the solvent. The NMR findings reported here are consistent with and amplify information on the stability of the trp-repressor obtained by other methods.

Amino Acid Sequence↗