Biomedical subjects

T R Schneider

Publications and source records attributed to T R Schneider.

At least 19 recordsLinked to original sources

PubMed2004

Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A.

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.

Acetates↗

PubMed2001

The effects of message framing and ethnic targeting on mammography use among low-income women.

The authors examined the effects that differently framed and targeted health messages have on persuading low-income women to obtain screening mammograms. The authors recruited 752 women over 40 years of age from community health clinics and public housing developments and assigned the women randomly to view videos that were either gain or loss framed and either targeted specifically to their ethnic groups or multicultural. Loss-framed, multicultural messages were most persuasive. The advantage of loss-framed, multicultural messages was especially apparent for Anglo women and Latinas but not for African American women. These effects were stronger after 6 months than after 12 months.

Adult↗

PubMed2001

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.

Acylation↗

PubMed2000

Ab initio structure determination of the lantibiotic mersacidin.

The crystal structure of mersacidin, a potential novel antibiotic against methicillin- and vancomycin-resistant Staphylococcus aureus strains, has been determined by ab initio methods. Despite all crystals being merohedrally twinned, an accurate structural model with an R value of 13.4% has been obtained at atomic resolution. With six molecules in the asymmetric unit and no atom heavier than sulfur, the structure corresponds to a protein of 120 amino acids and is the largest approximately equal-atom unknown structure solved by direct methods. In the crystal, the molecule assumes a compact fold different from that found by NMR in solution. Comparison of the NCS-related molecules reveals regions of variable flexibility. The region highly homologous to the related antibiotic actagardine is very rigid and possibly defines an essential building block of this class of new antibacterial substances.

Alanine↗

PubMed2000

Objective comparison of protein structures: error-scaled difference distance matrices.

Understanding of macromolecular function in many cases relies on the comparison of related structural models. Commonly used least-squares superposition methods suffer from bias introduced into the comparison process by the subjective choice of atoms employed for the superposition. Difference distance matrices are a more objective means of comparing structures as they do not depend on a particular superposition scheme. However, they suffer from very high noise originating from coordinate errors. Modern refinement programs allow the rigorous estimation of standard uncertainties for individual atomic positions. These errors can be propagated through the calculation of a difference distance matrix allowing one to assess the significance level of structural differences. An algorithm is presented which produces an intuitive graphical representation of difference distance matrices after normalization to their error levels. Two examples where its application was revealing are described. Alternatives are suggested for cases where rigorous estimation of individual errors by the inversion of the full least-squares matrix is not feasible. The method offers an unbiased way to detect significant similarities and differences between related structures, as encountered in studies of complexes and mutants or when multiple models are obtained from experiments such as crystal structures involving non-crystallographic symmetry or different crystal modifications, or ensembles derived from NMR spectroscopy.

Anti-Bacterial Agents↗

PubMed2000

Cardiovascular reactivity and adaptation to recurrent psychological stress: the moderating effects of evaluative observation.

The impact of evaluative observation on cardiovascular reactivity and adaptation to recurrent psychological stress was evaluated in 162 undergraduate men and women. All participants performed three mental arithmetic tasks with or without evaluative observation. Impedance cardiographic, blood pressure, task performance, and stress appraisal measures were recorded for each task. Evaluative observation moderated the effects of task repetition on cardiac reactivity but not vascular reactivity. The introduction of evaluative observation disrupted cardiac adaptation, resulting in a resurgence of beta-adrenergic cardiac reactivity (p < .005), whereas the removal of evaluative observation promoted cardiac adaptation. Evaluative observation also increased stress appraisals and slowed task performance. The results support the dual process theory of habituation, rather than stimulus comparator theory, but only partially support cognitive appraisal theory.

Adaptation, Psychological↗

PubMed1999

Influence of internal dynamics on accuracy of protein NMR structures: derivation of realistic model distance data from a long molecular dynamics trajectory.

In order to study the effect of internal dynamics on the accuracy of NMR structures in detail, we generated NOE distance data from a long molecular dynamics trajectory of BPTI. Cross-relaxation rates were calculated from the trajectory by analysis of the appropriate proton-proton vector autocorrelation functions. A criterion for the convergence of correlation functions was developed, and the analysis was restricted to those correlation functions that had converged within the simulation time. Effective distances were determined from the calculated cross-relaxation rates. Internal dynamics affected the derived distances in a realistic way, since they were subject both to radial averaging (which increases the cross-relaxation rate) and angular averaging (which decreases the cross-relaxation rate). The comparison of the effective distances with average distance between the protons during the trajectory showed that for most the effects of angular and distance averaging essentially cancel out. For these distances, the effective distance derived from an NOE is therefore a very good estimate of the average distance, or the distance in the average structure. However, for about 10% of the distances, the effective distance was more than 10% larger than the average distance, while for about 5%, it was more than 10% smaller, in some cases by more than 2 A. Little correlation is observed between the effects on cross-relaxation rates to different protons of the same residue. The results of this analysis have implications for the way structures are calculated from NOE distance data. For many distances, the assumption of a rigid structure is valid, and large error bounds would result in the loss of too much information content. On the other hand, the error bounds very often employed are not wide enough for some of the effects seen in our study.

Animals↗

PubMed1999

Persuasion for the purpose of cancer risk reduction: a discussion.

We comment on the preceding papers by Gerrard and Vernon concerning persuasion, perceived risk, and cancer-relevant behavior. Our purpose is to highlight several challenges for future investigators. First, relations between health cognition and health behavior (such as the link between perceived vulnerability and protective behaviors) are likely to be moderated by other variables, including individual differences and situational contexts. Second, we encourage health communication researchers to consider how persuasion is contextualized in social relationships and to employ mechanisms from the literature on social influence when promoting cancer prevention and early detection behaviors. Finally, we emphasize the importance of current feelings and anticipated emotions as motivators of salubrious actions.

Health Promotion↗

PubMed1999

1.7 A structure of the stabilized REIv mutant T39K. Application of local NCS restraints.

The X-ray structure of the T39K mutant of the variable domain of a human immunoglobulin kappa light chain has been determined at room temperature to 1.7 A resolution with a conventional R factor of 0. 182. T39K crystallizes in the triclinic space group P1 [a = 35.4 (1), b = 40.1 (1), c = 43.1 (1) A, alpha = 66.9 (1), beta = 85.4 (1), gamma = 73.8 (1) degrees ]. The unit-cell contains two monomers, related by a non-crystallographic twofold axis. The use of a novel type of local non-crystallographic symmetry restraints on related isotropic displacement parameters and 1-4 distances as incorporated in the refinement program SHELXL improves the model and quality of the maps, but local differences between both monomers in areas subject to different packing contacts can still be observed. 12 overall anisotropic scaling parameters were refined. These may have compensated for the difficulties in accurately scaling single rotation axis image plate data from a triclinic crystal, because of the scarcity of common equivalent reflections. The final model has been used to perform a number of tests on anisotropic scaling, non-crystallographic symmetry, anisotropic refinement, determination of standard uncertainties and bulk solvent correction. It is remarkable that removal of the NCS restraints from the final model caused Rfree to increase. These tests clarify the strategies for optimum use of SHELXL for refinement at medium as opposed to atomic resolution.

Crystallography, X-Ray↗

PubMed1999

Crystallization and preliminary X-ray analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine inhibitable) from Saccharomyces cerevisiae.

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (E.C. 4.1.2.15) catalyses the first step in the biosynthesis of aromatic amino acids: the condensation of phophoenolpyruvate and erythrose 4-phosphate to 3-deoxy-D-arabino-heptulosonate-7-phosphate. Diffraction-quality crystals of the tyrosine-inhibitable form of the enzyme from Saccharomyces cerevisiae have been obtained by the hanging-drop vapour-diffusion method in the presence of polyethylene glycol. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 81.5, b = 94.0, c = 104.6 A, alpha = 65.5, beta = 85.2, gamma = 75.0 degrees, and can be flash-cooled using glycerol as a cryoprotectant. A data set to 2.3 A has been collected at 120 K.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1999

Cardiovascular reactivity and adaptation to recurrent psychological stress: effects of prior task exposure.

The effects of prior task exposure on cardiovascular reactivity to stress were examined in two experiments by randomly assigning participants to repeated exposure groups that performed mental arithmetic pretest and test tasks versus delayed exposure groups that performed only the test task after prolonged rest. Impedance cardiographic and blood pressure measures were recorded continuously from 60 undergraduate men in Experiment 1 and 112 undergraduate men and women in Experiment 2. Task repetition attenuated cardiovascular reactivity and improved task performance in repeated exposure groups (p < .001), suggesting an integrated process of behavioral adaptation. During the test task, delayed exposure groups showed greater cardiac reactivity (p < .01), but not vascular reactivity, than repeated exposure groups. Thus, cardiac reactivity varied as a specific function of prior task exposure, whereas vascular reactivity varied as a general function of time.

Adaptation, Psychological↗

PubMed1998

Refinement of triclinic hen egg-white lysozyme at atomic resolution.

X-ray diffraction data have been collected at both low (120 K) and room temperature from triclinic crystals of hen egg-white lysozyme to 0.925 and 0.950 A resolution, respectively, using synchrotron radiation. Data from one crystal were sufficient for the low-temperature study, whereas three crystals were required at room temperature. Refinement was carried out using the programs PROLSQ, ARP and SHELXL to give final conventional R factors of 8.98 and 10.48% for data with F > 4sigma(F) for the low- and room-temperature structures, respectively. The estimated r.m.s. coordinate error is 0.032 A for protein atoms, 0.050 A for all atoms in the low-temperature study, and 0.038 A for protein atoms and 0.049 A for all atoms in the room-temperature case, as estimated from inversion of the blocked least-squares matrix. The low-temperature study revealed that the side chains of 24 amino acids had multiple conformations. A total of 250 waters, six nitrate ions and three acetate ions, two of which were modelled with alternate orientations were located in the electron-density maps. Three sections of the main chain were modelled in alternate conformations. The room-temperature study produced a model with multiple conformations for eight side chains and a total of 139 water molecules, six nitrate but no acetate ions. The occupancies of the water molecules were refined in both structures and this step was shown to be meaningful when assessed by use of the free R factor. A detailed description and comparison of the structures is made with reference to the previously reported structure refined at 2.0 A resolution.

Animals↗

PubMed1998

Loop closure and intersubunit communication in tryptophan synthase.

Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented. The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263,17857-17871] and Rhee et al. [(1997) Biochemistry 36, 7664-7680], we find that the presence of an alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site. Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alphaL2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.

Alanine↗

PubMed1998

Structure of balhimycin and its complex with solvent molecules.

Balhimycin is a naturally occurring glycopeptide antibiotic, related to vancomycin which acts by binding nascent bacterial cell-wall peptide ending in the sequence D-Ala-D-Ala. Crystals of balhimycin are monoclinic, space group P21, a = 20.48 (10), b = 43.93 (21), c = 27.76 (14) A, beta = 100.5 (5) degrees with four independent antibiotic molecules, three molecules of 2-methyl-2,4-pentanediol, two citrate ions, three acetate ions and 127.5 water molecules in the asymmetric unit. With an asymmetric unit larger than those of the smallest proteins and a solvent content of about 32%, the crystals have similar diffraction properties to those of small proteins. 27387 unique reflections were collected using synchrotron radiation. The structure was solved by a standard protein technique, the molecular-replacement method, using ureido-balhimycin as search model. The anisotropic refinement against all F2 data between 0.96 and 45 A converged to a conventional R value of 11.27% with R1= Sigma||Fo|-|Fc||/Sigma|Fo| for the 24623 data with I > 2sigma(I) and 12.58% for all 27387 data. The four monomers possess fairly similar conformations (r.m.s. deviation 0.7 A). Two antibiotic molecules form a tight dimer with antiparallel hydrogen bonds between the peptide backbone as well as between the vancosamine residues and the peptide backbone. In each of the two dimers, one binding pocket is occupied by a citrate ion and the other by an acetate ion. The dimer units are linked in the crystal by hydrogen bonds to form infinite chains.

Amino Acid Sequence↗

PubMed1998

The ensemble-averaged impedance cardiogram: an evaluation of scoring methods and interrater reliability.

The interrater reliability and concurrent validity of two methods of scoring the ensemble-averaged impedance cardiogram were evaluated. Impedance cardiographic and electrocardiographic signals were recorded from 40 undergraduate men and women during a baseline rest period and a vocal mental arithmetic task period. Recordings were scored by four rates using a conventional method, involving ensemble averaging after careful editing of beat-to-beat waveforms, and a streamlined method, involving ensemble averaging without beat-to-beat editing. Intraclass correlations for interrater reliability exceeded .92, whereas intraclass correlations for concurrent validity exceeded .97, indicating excellent agreement between rates and scoring methods for all cardiac measures. The streamlined method was significantly faster than the conventional method. The results indicate that variations in beat-to-beat editing do not constitute a serious source of error in the ensemble-averaged impedance cardiogram and support the interrater reliability and concurrent validity of the two scoring methods.

Adult↗

PubMed1998

A test of the validity of the method of constant stimuli as an index of heartbeat detection.

A heartbeat detection task, based on the method of constant stimuli (MCS), requires participants to judge which of a series of tones presented at different delays following a heartbeat are coincident with the heartbeat. Two experiments were conducted to test the validity of the MCS in assessing individual differences in the ability to detect heartbeat sensations. The first experiment found the MCS to be valid, in that about one-third of subjects met the criterion for classification as heartbeat detectors when the tones were presented with respect to their actual heartbeats, but only one subject met the criterion when the tones were presented with respect to previously recorded heartbeats. The second experiment manipulated the timing of the comparison intervals (i.e., R + 100-600 ms rather than R + 0-500 ms) to examine the validity of the median R-wave to tone interval as an index of temporal location of perceived heartbeats, and found it to be a valid index.

Acoustic Stimulation↗

PubMed1997

X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain.

BACKGROUND: Immunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation. Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation. Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts. RESULTS: Immunoglobulin REIv is the variable domain of a human kappa light chain. The disulfide-free variant REIv-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 A resolution). The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REIv-T39K. This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge. The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond. There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant. CONCLUSIONS: Immunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach. First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines. Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E. coli cell. Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.

Amino Acid Sequence↗

PubMed1996

Crystal structure of vancomycin.

BACKGROUND: Vancomycin and other related glycopeptide antibiotics are clinically very important because they often represent the last line of defence against bacteria that have developed resistance to antibiotics. Vancomycin is believed to act by binding nascent cell wall mucopeptides terminating in the sequence D-Ala-D-Ala, weakening the resulting cell wall. Extensive NMR and other studies have shown that the formation of asymmetric antibiotic dimers is important in peptide binding. Despite intensive efforts the crystal structure of vancomycin has been extremely difficult to obtain, partly because high-resolution data were unavailable, and partly because the structure was too large to be solved by conventional "direct methods'. RESULTS: Using low-temperature synchrotron X-ray data combined with new ab initio techniques for solving the crystallographic phase problem, we have succeeded in determining the crystal structure of vancomycin at atomic resolution. The structure provides much detailed information that should prove invaluable in modelling and mechanistic studies. CONCLUSIONS: Our structure confirms that vancomycin exists as an asymmetric dimer. The dimer conformation allows the docking of two D-Ala-D-Ala peptides in opposite directions; these presumably would be attached to different glycopeptide strands. In the crystal, one of the binding pockets is occupied by an acetate ion that mimics the C terminus of the nascent cell wall peptide; the other is closed by the asparagine sidechain, which occupies the place of a ligand. The occupied binding pocket exhibits high flexibility but the closed binding pocket is relatively rigid. We propose that the asparagine sidechain may hold the binding pocket in a suitable conformation for peptide docking, swinging out of the way when the peptide enters the binding pocket.

Anti-Bacterial Agents↗