Index of suspicion. Case #2. Diagnosis: California encephalitis.
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Child↗
Biomedical subjects
R S Keyes
Publications and source records attributed to R S Keyes.
Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels.
Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into duplexes of different sizes were found to display a helix length dependence and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on the dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry. 34:9265-9276). This sensitivity to size, which reflects global tumbling, is now reported for the more flexible five-atom-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into nucleic acids of different sizes. The DUAPs containing B-DNA systems were simulated with the same dynamic cylinder model, giving S = 0.20 +/- 0.01 for the more flexibly tethered spin label. This shows that S is dependent on tether length but not on global motion. An analysis with the same motional model of the B-Z transition in a (dG-dC)n polymer containing the five-atom-tethered six-membered ring cytidylate spin label (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry. 29:8522-8528) revealed an increase in S from 0.15 +/- 0.01 to 0.26 +/- 0.01 in response to the B- to Z-DNA transition. This indicates that S is not only sensitive to tether length, but also to conformational changes in DNA. Both the DUAP- and the DCAT-labeled systems were also simulated with a base disk model. From the DUAP spectral series, the perpendicular component of the correlation time tau perpendicular describing the spin-labeled base diffusion was found to be sensitive to global tumbling, confirming earlier results obtained with DUMTA. The DCAT polymer results demonstrated that tau perpendicular monitors a conformational change from B- to Z-DNA, indicating that tau perpendicular is also sensitive to local base dynamics. These results confirm that the dynamics of five-atom-tethered nitroxides are coupled to the nucleic acid dynamics and, as with two-atom-tethered spin labels, can be characterized by S and tau perpendicular. The analyses of both spin-labeled systems provide good evidence for spin-labeled base motions within double-stranded DNA occurring on the nanosecond time scale, and establish that both labels can be used to monitor changes in global tumbling and local order parameter due to variations in DNA conformation and protein-DNA interactions.
Base Sequence↗
Spectroscopic probe for the detection of local DNA bending at an AAA triplet.
Spectroscopic evidence of a DNA bend in solution is presented by analyzing model 15-mer duplexes spin-labeled with the five-atom-tethered nitroxide DUAP located in the major groove. Three 15-mers containing AATT with DUAP enzymatically incorporated into three different positions yielded nearly identical line shapes while a fourth 15-mer containing AAATT produced an EPR spectrum with significant additional line broadening. These results are interpreted according to the dynamic cylinder model where the DNA dynamics are decoupled into overall and internal contributions. It is shown that the AAATT sequence induces a change in the internal dynamics characterized by local ordering of DUAP. The increase in ordering evident in 15-mers containing AAATT rather than AATT suggests that the former sequence gives rise to a bend toward the major groove resulting in spatial restriction of the probe.
Base Sequence↗
Spin-labeled nucleotide mobility in the boundary of the EcoRI endonuclease binding site.
A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 +/- 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S = 0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.
Binding Sites↗
Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance.
Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.
Computer Simulation↗
Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA.
DNA motions consist of several components which couple, making their investigation difficult. This study describes an approach for obtaining dynamical information by EPR when spin-labeled nucleic acids are examined. The analysis is accomplished by implementing two motional models. The first model (i.e., dynamic cylinder model) views the spin-labeled helix as a diffusing cylinder containing internal dynamics which are characterized by an order parameter. The second model (i.e., base disk model) provides correlation times describing the diffusion of the spin-labeled base. In each model, the nitroxide motion consists of both global and internal contributions. Dynamic cylinder and base disk simulations of four duplexes containing nitroxides attached to thymidine by a two-atom tether (DUMTA)-(dT)7DUMTA-(dT)7.(dA)15, [(dT)7DUMTA(dT)7]2.(dA)30, [(dT)7DUMTA(dT)7]3.(dA)45, and [(dT)7DUMTA(dT)7]m.-(dA)n--demonstrate the useful application of this approach. From dynamic cylinder simulations, the order parameter for internal motions is found to be independent of the helix length (S = 0.32 +/- 0.01). Previous base disk simulations of a DNA 26mer and polymer labeled with a five-atom-tethered nitroxide seemed to indicate that tau perpendicular was only sensitive to internal dynamics. Results from base disk simulations of DUMTA-labeled DNA indicate that the perpendicular component of the base disk correlation time (tau perpendicular = 1.4-6.2 ns) is sensitive to global dynamics. Thus, tau perpendicular is a quantitative indicator of both internal and global dynamics. Comparison of the two models reveals that tau perpendicular infinity S2 tau rb, where tau rb represents the rigid-body diffusion of the DNA helix. This relationship between S and tau perpendicular provides a framework for studying conformational changes and size-dependent phenomena in spin-labeled nucleic acids. Application of the dynamic cylinder model to a B-Z transition generates distinct values of S for each of the conformations, indicating that Z-DNA is more rigid than B-DNA.
Base Sequence↗
A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and single-strand binding proteins.
An EPR competition formalism is developed which provides relative affinities of proteins for nucleic acids. Two models for analyzing protein-nucleic acid interactions, one assuming independent binding sites (Model 1) and the other considering site overlap (Model 2), are examined with respect to their validity and limitations. The models are employed to derive affinity ratio relationships which are used to calculate the relative affinities of gene 32, gene 5, and SSB proteins for various nucleic acids. It is determined that although Model 2 must be used when determining absolute binding constants, by taking the ratio of binding constants the site overlap becomes unimportant under conditions of moderate to high cooperativity and relatively small site size. This allows Model 1 to considerably simplify binding analyses. Both models are applied to the single-strand binding proteins of bacteriophage T4 gene 32, bacteriophage fd gene 5, and the Escherichia coli ssb gene, and the results are compared.
Allosteric Regulation↗
Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues.
Conformation detection and base dynamics of spin-labeled Z-DNA have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The two synthesized and characterized probes used in this study were C(5)-nitroxide-labeled 2'-deoxycytidine 5'-triphosphates, pppDCAT and pppDCAVAT, which serve as suitable substrates for Micrococcus luteus DNA polymerase. Enzymatic incorporation of these probes into (dG-dC)n yields the EPR-active alternating copolymers (dG-dC,DCAT)n and (dG-dC,-DCAVAT)n. These polymers assume typical B- and Z-DNA conformations under respective low (0.1 M NaCl) and high (4.5 M NaCl) salt conditions, as evidenced by their UV-circular dichroism spectra. The EPR line shape of (dG-dC,DCAT)n in Z-form is unique and significantly different from the B-form EPR spectrum. A similar observation is made for (dG-dC,DCAVAT)n. Thus, the EPR line shapes of these spin-labeled DNAs are indicative of their local conformations. The EPR spectra, analyzed with a previously published motional model [Kao, S.-C., Polnaszek, C.F., Toppin, C.R., & Bobst, A.M. (1983) Biochemistry 22, 5563-5568], indicate tau perpendicular values of 4 and 7 ns for the B- and Z-forms, respectively. Therefore, the base dynamics of Z-DNA are about two times slower than in B-DNA.
Circular Dichroism↗
An electron paramagnetic resonance probe to detect local Z-DNA conformations.
We present spectroscopic evidence for an electron paramagnetic resonance (EPR) probe to detect local Z-DNA conformations in synthetic DNA. A spin labeled deoxycytidine-5'-triphosphate (pppDCAT) was co-polymerized with Micrococcus luteus DNA polymerase to yield the spin active alternating co-polymer (dG-dC,DCAT)n. The EPR spectrum of (dG-dC,DCAT)n in the Z-DNA conformation indicates a decrease in the local base dynamics by about a factor of two as compared to that computed for B-DNA. A control experiment conducted with (dA-dT, DUAT)n under similar salt conditions rules out the possibility of observing salt induced artifacts.
Base Sequence↗
Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease.
Deoxyuridine analogs spin labeled in position 5 have been enzymatically incorporated sequence specifically into an oligodeoxyribonucleotide to form a spin-labeled 26-mer. The 26-mer contains the EcoRI-binding site and two labels which are located symmetrically close to the binding site. The labels are separated from one another far beyond the Heisenberg spin-exchange distance. The local base motion as determined by ESR spectroscopy is of the order of 4 ns in the oligonucleotide duplex. This is the same value as reported earlier for local T motions in polynucleotide duplexes, thereby providing direct experimental evidence that the ESR line shape of spin levels covalently attached to nucleic acids depends primarily on the local dynamics of the nucleic acid building blocks.
Affinity Labels↗
Function of the pericardium and pericardioperitoneal canal in elasmobranch fishes.
Previous studies of cardiac function in elasmobranch fishes have not included the influence of the pericardioperitoneal canal on pericardial pressure and volume and thus on cardiac function. Accordingly, we studied the function of the pericardium and pericardioperitoneal canal in sharks and rays. We found negative pericardial pressure that rose to a plateau of approximately 0 mmHg when fluid was infused into the pericardium with the canal undisturbed. However, this pericardial pressure elevation caused severe cardiac tamponade. After the canal was occluded, the pressure plateau was substituted with an exponential rise. We injected radioisotopes into the pericardial cavity and obtained scintigrams several hours later. The scans and counts of body fluids and tissues indicated absorption, disputing the suggestion that the primary function of the canal may be inadequate absorption of pericardial fluid. We conclude that the pericardioperitoneal canal maintains negative pericardial pressure, which is a prerequisite in elasmobranch fishes and may serve to regulate pericardial pressure level to optimize cardiac function in relation to changes in cardiac size.
Absorption↗