Biomedical subjects

M S Searle

Publications and source records attributed to M S Searle.

At least 37 records · Page 2Linked to original sources

PubMed1998

Prion protein fragments spanning helix 1 and both strands of beta sheet (residues 125-170) show evidence for predominantly helical propensity by CD and NMR.

BACKGROUND: Transmissible spongiform encephalopathies are a group of neurodegenerative disorders of man and animals that are believed to be caused by an alpha-helical to beta-sheet conformational change in the prion protein, PrP. Recently determined NMR structures of recombinant PrP (residues 121-231 and 90-231) have identified a short two-stranded anti-parallel beta sheet in the normal cellular form of the protein (PrPC). This beta sheet has been suggested to be involved in seeding the conformational transition to the disease-associated form (PrPSc) via a partially unfolded intermediate state. RESULTS: We describe CD and NMR studies of three peptides (125-170, 142-170 and 156-170) that span the beta-sheet and helix 1 region of PrP, forming a large part of the putative PrPSc-PrPC binding site that has been proposed to be important for self-seeding replication of PrPSc. The data suggest that all three peptides in water have predominantly helical propensities, which are enhanced in aqueous methanol (as judged by deviations from random-coil Halpha chemical shifts and 3JHalpha-NH values). Although the helical propensity is most marked in the region corresponding to helix 1 (144-154), it is also apparent for residues spanning the two beta-strand sequences. CONCLUSIONS: We have attempted to model the conformational properties of a partially unfolded state of PrP using peptide fragments spanning the region 125-170. We find no evidence in the sequence for any intrinsic conformational preference for the formation of extended beta-like structure that might be involved in promoting the PrPC-PrPSc conformational transition.

Animals↗

PubMed1997

The pattern of change in leisure activity behavior among older adults with arthritis.

This study employed a "ceasing participation" framework to examine changing leisure activity patterns. Respondents of the Living With Arthritis project were classified into four participation pattern categories. Results confirmed that older adults with arthritis are more likely to experience changes to their activity regimen than older adults without arthritis. A multi-group discriminant function analysis showed that arthritis severity distinguished those who tend to cease activity. Social network and age best distinguished those who quit activities without replacement. Results are placed in the context of coping strategies. Those who do not replace forfeited activities with other activities are least flexible in their response to their chronic condition and face challenges to their well-being.

Aged↗

PubMed1997

A new formula for population-based estimation of whole body muscle mass in males.

A new equation to estimate muscle mass in males was developed using parameters common to the 1981 Canada Fitness Survey and the male cadaver data of Martin et al. (1990b). The cadavers (N = 12) were randomly divided into two groups. The equation was developed on cadaver Group A and then validated on Group B. Once the equation with the most suitable variables was validated on Group B, it was redeveloped on combined data from Groups A and B. The final equation is as follows: muscle mass (gm) = Ht (0.031MUThG2 + 0.064CCG2 + 0.089CAG2) - 3,006; adjusted R2 = .96, SEE = 1,488 gm, F = 87.5, p = .0001. Variables (in cm) were Ht, height; MUThG, modified upper thigh girth; CCG, corrected calf girth; and CAG, corrected arm girth. The predictive ability of this equation was comparable to the original equation of Martin et al. (1990b) and can be a valuable tool for muscle mass estimation of male subjects in the 1981 Canada Fitness Survey.

Aged↗

PubMed1996

Native-like beta-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues.

The conformational properties of protein fragments have been widely studied as models of the earliest initiation events in protein folding. While native-like alpha-helices and beta-turns have been identified, less is known about the factors that underly beta-sheet formation, in particular beta-hairpins, where considerably greater long-range order is required. The N-terminal 20 residue sequence of native ferredoxin I (from the blue-green alga Aphanothece sacrum) forms a beta-hairpin in the native structure and has been studied in isolation by NMR and CD spectroscopy. Local native-like interactions alone are unable to stabilize significantly a folded conformation of the 20-residue fragment in purely aqueous solution. However, we show that the addition of low levels of organic co-solvents promotes formation of native-like beta-hairpin structure. The results suggest an intrinsic propensity of the peptide to form a native-like beta-hairpin structure, and that the organic co-solvent acts in lieu of the stabilizing influence of tertiary interactions (probably hydrophobic contacts) which occur in the folding of the complete ferredoxin sequence. The structure of the isolated hairpin, including the native-like register of interstrand hydrogen bonding interactions, appears to be determined entirely by the amino acid sequence. The solvent conditions employed have enabled this intrinsic property to be established.

Amino Acid Sequence↗

PubMed1995

A short linear peptide derived from the N-terminal sequence of ubiquitin folds into a water-stable non-native beta-hairpin.

A 16-residue peptide derived from the N-terminal sequence of ubiquitin forms a stable monomeric beta-hairpin that is estimated to be approximately 80% populated in aqueous solution. The peptide sequence has been modified from native ubiquitin by replacing the five residues found in a type I G1 bulged turn (Thr-Leu-Thr-Gly-Lys) with four residues (Asn-Pro-Asp-Gly) to maximize the probability of forming a beta-turn. Unexpectedly, the bulged turn conformation is re-established in the beta-hairpin in solution with two consequences: a one-amino acid frameshift in the alignment of the peptide main chain occurs relative to the native hairpin, and side chains formerly on opposite faces of the hairpin are brought together on the same face. The presence of the bulged turn in native ubiquitin may help in the avoidance of the stable non-native register of amino acids found here which would be unproductive for folding.

Amino Acid Sequence↗

PubMed1995

Activity participation and well-being among older people with arthritis.

This study tests the hypotheses that (a) severity of arthritis is inversely associated with frequency of activity participation, and (b) arthritis sufferers who maintain higher levels of participation, particularly in activities which are social in nature, are less likely to experience a decline in well-being. Three activity types are considered: social, physical, and solitary. Results indicate that well-being is influenced by social activity, whereas solitary and physical activity have minimal impact. This suggests that elders with arthritis need not remove themselves from the pursuit of activity and should be encouraged to develop new interests when physical functioning fails. The study also demonstrates the utility of considering activity as multidimensional.

Aged↗

PubMed1995

Child abuse and neglect by parents with disabilities: A tale of two families.

Two families, in which the children had been placed in foster care due to abuse and neglect by parents who had disabilities, were studied. In the first case, the mother was instructed in skills that our assessment suggested were important for her child's survival. The mother readily acquired and applied these skills, a fact reflected both in changes in her behavior and in changes in the child's well-being. In the second case, the parent's incremental resumption of child custody was made contingent upon completion of relevant parenting tasks. Initially, improvements in the completion of such tasks were evident, but over time and with the onset of militating factors, no further progress was made and all parental rights were terminated. The implications of these cases for behavior analysis and the effort to reunite and preserve families are discussed.

Journal Article↗

PubMed1994

Binding of quinomycin antibiotic UK-65,662 to DNA: 1H-n.m.r. studies of drug-induced changes in DNA conformation in complexes with d(ACGT)2 and d(GACGTC)2.

Quinomycin antibiotic UK-65,662 binds selectively to the 5'-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5'-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.

Anti-Bacterial Agents↗

PubMed1994

The structure of an asymmetric dimer relevant to the mode of action of the glycopeptide antibiotics.

BACKGROUND: Glycopeptide antibiotics of the vancomycin group are of crucial clinical importance in the treatment of methicillin resistant Staphylococcus aureus (MRSA)--the often lethal 'super-bug'--characterized by its resistance to a wide range of antibiotics in common use. The antibiotics exert their physiological action by blocking cell wall synthesis through recognition of nascent cell wall mucopeptides terminating in the sequence -D-Ala-D-Ala. Evidence suggests that the antibiotics are able to enhance their biological activity by the formation of homodimers, and this is supported by the observation that dimerization and peptide binding in vitro are cooperative phenomena. The basis of this enhancement is not understood at the molecular level. RESULTS: The first detailed structure of a dimeric glycopeptide antibiotic, that of eremomycin, is presented based upon solution NMR data. The overall structure of the dimer complex is asymmetric. The source of this asymmetry--a parallel alignment and mutual interaction of the disaccharides--appears to promote dimerization through specific sugar-sugar recognition. CONCLUSIONS: A molecular basis for the observed cooperativity of cell wall peptide binding by eremomycin is evident from these studies of the dimer. The carboxylate anion of the cell wall component, which is crucial to binding, forms an amide-mediated ion-pair interaction to the alkylammonium ion of the ring 6 sugar in the other half of the dimer making the structure and positioning of this sugar important in mediating cooperativity.

Amino Acid Sequence↗

PubMed1993

On the stability of nucleic acid structures in solution: enthalpy-entropy compensations, internal rotations and reversibility.

The thermodynamics of self-association (stacking) of free bases and nucleotides, intramolecular stacking in dinucleotides, nearest-neighbour base pair stacking interactions in duplex DNA and RNA, and the formation of hairpin loops illustrate enthalpy/entropy compensations. Large stacking exothermicities are associated with large negative entropy changes that ensure that delta G is small, permitting readily reversible associations in solution. We rationalise enthalpy/entropy compensations with reference to residual motions and torsional vibrations which make a larger entropic contribution to binding when - delta H approximately kT (thermal energy at room temperature), than when - delta H >> kT. We present a factorisation of experimental free energies for helix formation in terms of approximate contributions from the restriction of rotations, hydrophobic interactions, electrostatic interactions due to base stacking, and contributions from hydrogen bonding, and estimate the adverse free energy cost per rotor (mainly entropy) of ordering the phosphate backbone as between 1.9 and 5.4 kJ mol-1 [averaged over 12 rotors per base pair for A-U on A-U stacking (lower limit), and G-C on C-G stacking (upper limit)]. The largest cost is associated with the most exothermic stacking interactions, while the range of values is consistent with earlier conclusions from data on the fusion of hydrocarbon chains (lower value), and with entropy changes in covalent isomerisations of small molecules involving severe restrictions (upper value).

Base Composition↗

PubMed1993

Toward an estimation of binding constants in aqueous solution: studies of associations of vancomycin group antibiotics.

An approach toward the estimation of binding constants for organic molecules in aqueous solution is presented, based upon a partitioning of the free energy of binding. Consideration is given to polar and hydrophobic contributions and to the entropic cost of rotor restrictions and bimolecular associations. Several parameters (derived from an analysis of entropy changes upon the melting of crystals and from the binding of cell wall peptide analogues to the antibiotic ristocetin A) which may be useful guides to a crude understanding of binding phenomena are presented: (i) amide-amide hydrogen bond strengths of -(1 to 7) +/- 2 kJ.mol-1, (ii) a hydrophobic effect of -0.2 +/- 0.05 kJ.mol-1.A-2 of hydrocarbon removed from exposure to water in the binding process, and (iii) free energy costs for rotor restrictions of 3.5-5.0 kJ.mol-1. The validity of the parameters for hydrogen bond strengths is dependent on the validity of the other two parameters. The phenomenon of entropy/enthalpy compensation is considered, with the conclusion that enthalpic barriers to dissociations will result in larger losses in translational and rotational entropy in the association step. The dimerization of some vancomycin group antibiotics is strongly exothermic (-36 to -51 kJ.mol-1) and is promoted by a factor of 50-100 by a disaccharide attached to ring 4 (in vancomycin and eremomycin) and by a factor of ca. 1000 by an amino-sugar attached to the benzylic position of ring 6 in eremomycin. The dimerization process (which, as required for an exothermic association, appears to be costly in entropy) may be relevant to the mode of action of the antibiotics.

Anti-Bacterial Agents↗

PubMed1993

Interaction of Hoechst 33258 with the minor groove of the A + T-rich DNA duplex d(GGTAATTACC)2 studied in solution by NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the A + T-rich DNA duplex d(GGTAATTACC)2 has been investigated in solution by two-dimensional 1H-NMR spectroscopy. Many intermolecular NOEs define the position and orientation of the Hoechst molecule at the centre of the duplex, binding to a single site located within the minor groove of the AATT sequence. This is despite the opportunity for Hoechst 33258 to bind to as many as five unique sites containing the minimum requirement of three consecutive A.T base pairs. All intermolecular NOEs are detected between protons on the concave edge of Hoechst, many with the adenine H2s on the floor of the minor groove. The NOEs are consistent with interproton distances measured within a partially restrained energy-minimised structure of the complex. The AATT sequence provides the key recognition features for tight binding, including a particularly narrow minor groove, with no evidence for the partial G.C specificity previously proposed to be necessary to accommodate the bulky N-methylpiperazine ring. While chemical exchange cross-peaks between symmetry-related adenine H2s on opposite strands of the duplex identify a very slow rate of dissociation of the drug from the complex at 298 K (exchange rate approximately 1.2 s-1). Averaging of chemical shifts in the phenyl ring of Hoechst 33258 are indicative of rapid ring-flipping motions in the bound state.

Adenine↗

PubMed1992

Interaction of the anthracycline antibiotic nogalamycin with the hexamer duplex d(5'-GACGTC)2. An NMR and molecular modelling study.

The interaction of the anthracycline antibiotic nogalamycin with the single 5'-CpG binding site at the centre of the hexamer duplex d(GACGTC)2 has been investigated in solution by NMR spectroscopy. 41 NOE constrains between antibiotic and nucleotide protons provide input parameters for the determination of a family of conformers computed using energy minimisation and restrained molecular dynamics. Nogalamycin adopts a 'threaded' binding orientation in which the drug chromophore passes through the DNA helix positioning the nogalose sugar in the minor groove and the positively charged bicycloamino sugar in the major groove. The complex is stabilised by hydrogen bonding, electrostatic and van der Waals interactions. In the major groove hydrogen bonding interactions are identified between the 2'-OH and 4'-OH groups, on the rigidly oriented bicyclo sugar, and the guanine N7 and cytosine 4-NH2 of one G.C base pair and appear to account for the requirement for a G.C base pair at the intercalation site. In the minor groove we identify a hydrogen bond between the carbonyl oxygen of the methyl ester group on ring A and the 2-NH2 group of guanine at the intercalation site. The nogalose and ring A form a continuous domain that interacts through van der Waals contacts with the floor and walls of the minor groove over a span of four basepairs, d(GACGTC)2. The NOE data together with energy-minimised structures reveal distortions of the DNA conformation involving a total helix unwinding of about 16 degrees and buckling of the G.C base pairs by about 22 degrees and -9 degrees at the intercalation site. The base pairs effectively wrap around the drug chromophore optimising van der Waals contacts between the two and minimising cavities in the structure. The solution data also reveal that the bound antibiotic substantially slows the rate of exchange of the imino protons of not only the G.C base pairs forming the intercalation site but also that of an A.T base pair which is occluded by the sugar moieties lying in each groove. The data present an opportunity for comparison with two crystal structures of nogalamycin-DNA complexes in which ligand molecules are bound at terminal 5'-CpG sites.

Binding Sites↗

PubMed1992

31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5'-GCATGC)2 in its complex with the antibiotic nogalamycin.

Heteronuclear chemical shift correlation experiments confirm that the two down-field shifted 31P resonances in the spectrum of the (nogalamycin)2-d(GCATGC)2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R1, R2 and [1H]-31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3'-31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.

DNA↗

PubMed1991

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.

Anthracyclines↗

PubMed1991

Structural analysis of the decanucleotide duplex d(GGTAATTACC)2 using 1H NMR spectroscopy.

The conformation of the decanucleotide duplex d(GGTAATTACC)2 has been investigated in solution by one- and two-dimensional proton NMR spectroscopy. Intra- and inter-nucleotide two-dimensional nuclear Overhauser enhancement data, recorded at mixing times between 15 and 250 ms, reveal a right-handed B-DNA structure. The data also show that the A-T basepairs of the TAATTA tract are highly propeller twisted and the minor groove is particularly narrow.

Base Sequence↗

PubMed1990

Hoogsteen versus Watson-Crick A-T basepairing in DNA complexes of a new group of 'quinomycin-like' antibiotics.

The interaction of a new group of 'quinomycin-like' antibiotics with the DNA duplexes d(ACGT)2 and d(GACGTC)2 has been investigated in solution by 1H NMR spectroscopy. By monitoring the intensity of intranucleotide base H6/H8 to deoxyribose H1'NOE cross-peaks we conclude that the terminal A-T basepairs flanking the CG bisintercalation site in the d(ACGT)2 complex adopt the Hoogsteen bonding scheme, with the purine base in a syn conformation. By comparison in the d(GACGTC)2 complex all glycosidic bond angles are anti, consistent with a preferred Watson-Crick basepairing scheme. Both DNA duplexes appear to be significantly unwound compared with the ligand-free DNAs. The data illustrate the influence of helical constraints on the stability of the Hoogsteen bonding scheme adjacent to the drug binding sites.

Anti-Bacterial Agents↗

PubMed1990

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5'-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.

Base Sequence↗