Biomedical subjects

C H Arrowsmith

Publications and source records attributed to C H Arrowsmith.

52 records · Page 3Linked to original sources

PubMed1998

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: a role for N-terminal residues in tandem binding.

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H, 15N, 13C labelled intact and (des2-7) trp repressor (delta 2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound delta 2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.

Amino Acid Sequence↗

PubMed1998

New perceptions of transcription factor properties from NMR.

The complementarity of NMR and X-ray crystallography for biomacromolecular studies has been particularly evident in analysis of transcription factor structures and interactions. While X-ray crystallography can be used to tackle relatively complicated structural problems including multicomponent (three and higher) complexes, NMR studies have provided new insights into the nature of protein-DNA and protein-protein interactions that would be difficult to obtain by other biophysical methods. We describe herein some of the novel and important information recently derived from NMR studies of transcription factors.

Animals↗

PubMed1997

ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging.

Alleles↗

PubMed1997

Quantitative determination of conformational, dynamic, and kinetic parameters of a ligand-protein/DNA complex from a complete relaxation and conformational exchange matrix analysis of intermolecular transferred NOESY.

We report a quantitative analysis of the 13C-edited intermolecular transferred NOESY (inter-TrNOESY) spectrum of the trp-repressor/operator complex (trp-rep/op) with [ul-13C/15N]-L-tryptophan corepressor using a computer program implementing complete relaxation and conformational exchange matrix (CORCEMA) methodology [Moseley et al. (1995) J. Magn. Reson. 108B, 243-261]. Using complete mixing time curves of three inter-TrNOESY peaks between the tryptophan and the Trp-rep/op, this self-consistent analysis determined the correlation time of the bound species (tauB = 13.5 ns) and the exchange off-rate (k(off) = 3.6 s(-1)) of the corepressor. In addition, the analysis estimated the correlation time of the free species (tauF approximately 0.15 ns). Also, we demonstrate the sensitivity of these inter-TrNOESY peaks to several factors including the k(off) and orientation of the tryptophan corepressor within the binding site. The analysis indicates that the crystal structure orientation for the corepressor is compatible with the solution NMR data.

Bacterial Proteins↗

PubMed1996

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Fungal Proteins↗

PubMed1996

New insights into p53 function from structural studies.

Recent structural analysis of p53 has greatly enhanced our understanding of the biochemical activities of this protein by presenting us with a detailed picture of the chemical groups in the protein that are involved in protein stability, conformation and functional interactions. The current structures form the basis for the design of potential therapeutics which could, for example, revert a DNA-binding mutant back to a DNA-binding competent conformation. The structure of the tet domain forms the basis for designing an active therapeutic p53 with an oligomerization domain which would not cross react with a DNA-binding mutant p53. However, as useful as these structures have been in providing insight into the structure/function relationship for p53, a complete understanding of this protein awaits more detailed information on the full-length protein. In this respect, one of the most useful roles for future structural studies will be to help identify the nature of the conformational transition between latent and active p53, and how it can be modulated.

Base Sequence↗

PubMed1995

Structural analysis and comparison of the C-terminal transport signal domains of hemolysin A and leukotoxin A.

NMR spectroscopy was used to study the structure of the C-terminal signal sequences of the bacterial toxins, hemolysin A(HlyA) and leukotoxin A (LktA). The two signals share little sequence homology; however, both can direct toxin transport with equal efficiency. We report here that in a membrane mimetic environment both peptides form two short non-interacting alpha-helices separated by a short loop. This higher order structure may be a common feature of C-terminal signals and may be required for interaction with the membrane associated transporter complex.

Amino Acid Sequence↗

PubMed1995

Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor.

The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N4<==>4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 microM have a transition temperature (Tm) of 75.3 degrees C while at 0.5 microM the value drops to 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)-1 with a heat capacity change of 387 cal (K.mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

PubMed1995

Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.

The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

Bacterial Proteins↗

PubMed1995

Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.

[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Bacterial Proteins↗

PubMed1994

Solution structure of the tetrameric minimum transforming domain of p53.

We report the solution structure of the minimum transforming domain (residues 303-366) of human p53 (p53tet) determined by multidimensional NMR spectroscopy. This domain contains a number of important functions associated with p53 activity including transformation, oligomerization, nuclear localization and a phosphorylation site for p34/cdc2 kinase. p53tet forms a symmetric dimer of dimers that is significantly different from a recent structure reported for a shorter construct of this domain. Phosphorylation of Ser 315 has only minor structural consequences, as this region of the protein is unstructured. Modelling based on the p53tet structure suggests possible modes of interaction between adjacent domains in full-length p53 as well as modes of interaction with DNA.

Amino Acid Sequence↗

PubMed1993

Refined solution structures of the Escherichia coli trp holo- and aporepressor.

The solution structures of the trp-repressor from Escherichia coli in both the liganded (holo-) and unliganded (apo-) form, have been refined by restrained molecular dynamics with simulated annealing using the program XPLOR and additional experimental constraints. The ensemble of refined holorepressor structures have a root-mean-square deviation (r.m.s.d.) of 0.8 A relative to the average structure for the backbone of the dimer core (helices A, B, C, A', B', C') and 2.5 A for the helix-turn-helix DNA-binding domain (helices D and E). The corresponding values for the aporepressor are 0.9 A for the backbone of the ABC-dimer core and 3.2 A for the DE helix-turn-helix. The r.m.s.d. of the average structures from the corresponding crystal structures are 2.3 A for the holorepressor ABC core and 4.2 A for its DE region; 2.3 A for the aporepressor core and 5.5 A for its DE region. The relative disorder of the DNA-binding domain is reflected in a number of experimental parameters including substantially more rapid backbone proton exchange rates, exchange-limited relaxation times and crystallographic B-factors. The stabilizing effect of the L-Trp ligand is evident in these measurements, as it is in the higher precision of the holorepressor structure.

Amino Acid Sequence↗

PubMed1992

The effect of selective deuteration on magnetization transfer in larger proteins.

The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of the E. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuterated trp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NHi-NHi+1 cross peaks in the alpha-helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.

Amino Acid Sequence↗

PubMed1990

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor.

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.

Amino Acid Sequence↗

PubMed1989

NMR studies of the Escherichia coli trp aporepressor. Sequence-specific assignment of the aromatic proton resonances.

The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z. & Sigler, P.B. (1987) Nature 327, 591-597]. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.

Amino Acid Sequence↗

PubMed1989

NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding.

The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism. The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared. Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region. The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms". Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy. Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region. DNA binding by the armless protein does not reduce the mobility of these residues. Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy. These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.

Bacterial Proteins↗