Ultrafast time-resolved crystallography.
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Animals↗
Biomedical subjects
K Moffat
Publications and source records attributed to K Moffat.
Initial trajectory of carbon monoxide after photodissociation from myoglobin at cryogenic temperatures.
Migration of the CO ligand following photolysis of carbonmonoxy myoglobin (MbCO) in single crystals has been investigated by time-resolved X-ray diffraction at 40K. After short illumination by weak visible light at a photolysis rate of approximately 1 s-1, the photodissociated CO molecule is found about 1 A from its bound location. After continuous illumination over several hours, the CO molecule migrates to a more distant site in the distal pocket, about 2.5 A from its bound location. Migration of the ligand under continuous illumination accounts for different locations of the photodissociated CO molecule previously reported in three cryocrystallographic studies [Teng, T.-Y., et al. (1994) Nat. Struct. Biol. 1, 701-705; Schlichting, I., et al. (1994) Nature 371, 808-812; Hartmann, H., et al. (1996) Proc.Natl.Acad. Sci. U.S.A. 93, 7013-7016]. Due to the different photolysis protocols employed in these studies, each reveals a different part of the trajectory of the photodissociated CO molecule. When the different experimental parts of the trajectory at 40 K are pieced together and combined with our nanosecond time-resolved studies at room temperature [Srajer, V., et al. (1996) Science 274, 1726-1729], excellent agreement is obtained with recent theoretical predictions of the CO probability distribution in the ligand pocket [Vitkup, D., et al. (1997) Nat. Struct. Biol. 4, 202-208]. The heme relaxation that accompanies ligand photolysis is incomplete, about 30% of that associated with the conversion of MbCO to deoxy-Mb at room temperature, and independent of the duration of illumination. Other tertiary structural changes in the globin are also greatly diminished. The globin structure is therefore very rigid at cryogenic temperatures, and structural relaxation is greatly hindered, consistent with numerous spectroscopic measurements.
Carbon Monoxide↗
Structure of a protein photocycle intermediate by millisecond time-resolved crystallography.
The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.
Bacterial Proteins↗
Comparison of 5-mg and 10-mg loading doses in initiation of warfarin therapy.
BACKGROUND: Loading doses of warfarin that are larger than those used for maintenance therapy are widely used in clinical practice, but they have never been prospectively evaluated. OBJECTIVE: To compare the effect of 5- and 10-mg loading doses of warfarin on laboratory markers of warfarin's anticoagulant effect. DESIGN: Randomized clinical trial. SETTING: Tertiary care teaching hospital. PATIENTS: 49 patients seen over a 5-month period with a target international normalized ratio (INR) of 2.0 to 3.0. INTERVENTION: Patients were randomly assigned to receive an initial dose of 5 or 10 mg of warfarin. Subsequent doses of warfarin were administered on the basis of dosing nomograms. MEASUREMENTS: INRs and levels of factors II, VII, IX, and X and protein C were measured daily for 5 days. RESULTS: 11 of 25 patients in the 10-mg group (44% [95% CI, 34% to 54%]) and 2 of 24 patients in the 5-mg group (8% [CI, 3% to 14%]) had INRs greater than 2.0 at 36 hours (P = 0.005), at which time the factor VII levels were 27% (CI, 18% to 36%) in the 10-mg group and 54% (CI, 43% to 65%) in the 5-mg group (P < 0.001). In contrast, factor II levels were 74% (CI, 67% to 81%) in the 10-mg group and 82% (CI, 73% to 93%) in the 5-mg group (P > 0.2). At 60 hours, 9 of 25 patients in the 10-mg group (36% [CI, 17% to 54%]) and no patients in the 5-mg group had INRs greater than 3.0. At 84 hours, 15 of 24 patients in the 10-mg group (63% [CI, 43% to 81%]) and 19 of 24 patients in the 5-mg group (79% [CI, 62% to 95%]) had INRs between 2.0 and 3.0. Four patients in the 10-mg group and 1 patient in the 5-mg group received vitamin K for excessive prolongation of the INR. CONCLUSIONS: A 5-mg loading dose of warfarin produces less excess anticoagulation than does a 10-mg loading dose; the smaller dose also avoids the development of a potential hypercoagulable state caused by precipitous decreases in levels of protein C during the first 36 hours of warfarin therapy.
Anticoagulants↗
Laue diffraction.
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Crystallography, X-Ray↗
Synchrotron radiation applications to macromolecular crystallography.
Progress has been rapid in the development and application of four different types of macromolecular crystallographic experiment at synchrotron hard X-ray sources: multiwavelength anomalous diffraction; studies of crystals with very large unit cell dimensions; structure determination at atomic or near-atomic resolution; and time-resolved studies. The results illustrate the interplay between the advanced technical capabilities available at new beamlines and more challenging scientific issues.
Crystallography, X-Ray↗
Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography.
The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.
Carbon Monoxide↗
Insights into specificity of cleavage and mechanism of cell entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin.
BACKGROUND: Restriction, a highly specific ribotoxin made by the fungus Aspergillus restrictus, cleaves a single phosphodiester bond in the 28S RNA of eukaryotic ribosomes, inhibiting protein synthesis. The sequence around this cleavage site is a binding site for elongation factors, and is conserved in all cytoplasmic ribosomes. The catalytic mechanism of restrictocin and the reasons for its high substrate specificity are unknown. No structure has been determined for any other member of the Aspergillus ribotoxin family. RESULTS: The crystal structure of restrictocin was determined at 2.1 A resolution by single isomorphous replacement and anomalous scattering techniques, and refined to 1.7 A resolution using synchrotron Laue data. The structural core of the protein, in which a three-turn alpha helix is packed against a five-stranded antiparallel beta sheet, can be well aligned with that of ribonuclease T1. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA binding. CONCLUSIONS: Restriction appears to combine the catalytic components of T1 ribonucleases with the base recognition components of Sa ribonucleases. Modeling studies using an NMR structure of an RNA substrate analog suggest that the tertiary structure of the substrate RNA is important in protein-RNA recognition, fitting closely into the concavity of the presumed binding site. We speculate that the large 39-residue loop L3, which has similarities to loops found in lectin sugar-binding domains, may be responsible for restrictocin's ability to cross cell membranes.
Allergens↗
Feasibility and Realization of Single-Pulse Laue Diffraction on Macromolecular Crystals at ESRF.
Laue diffraction patterns with an exposure time of ca 60 ps have been acquired at the European Synchrotron Radiation Facility (ESRF) on protein crystals by using the single-bunch mode of the storage ring. A 10 ns laser pulse initiating photodissociation was synchronized with the X-ray pulse. The potential for a quantitative detection of conformational changes in proteins on the nanosecond timescale with this technique is demonstrated using the example of carbonmonoxymyoglobin, from simulations and real data. The instrumental aspects of the experiment (highly intense X-ray beam, fast shutter system, Laue camera, detector, laser apparatus and synchronization technique) are emphasized.
Journal Article↗
Reliability of the international normalized ratio for monitoring the induction phase of warfarin: comparison with the prothrombin time ratio.
The International Normalized Ratio (INR) was introduced to reduce the variability of prothrombin time (PT) reporting. One potential problem with the use of the INR is the assumption that its reliability is reduced when it is used to monitor patients during the induction phase of treatment. This shortcoming arises because the model used to establish the INR system is based on the use of pooled plasma from patients stabilized on warfarin for at least 6 weeks. Because the prolongation of the PT by warfarin during the induction phase mainly reflects reduction in factor VII levels (whereas the prolongation of the PT after 6 weeks of stabilization reflects reductions in factors X, II, and VII), there exists a potential for loss of accuracy of the INR during warfarin induction. To overcome this potential problem, it has been suggested that the PT ratio should be used to report results during the induction phase of treatment and that the INR system should be reserved for reporting results after the patient has been stabilized. This approach is confusing to the clinician. In addition, the validity of this approach has never been demonstrated in a clinical study. To address this issue, we studied 43 patients for the first 5 days after they started warfarin therapy. We measured the PT in the same plasma samples from each patient with five different commercial thromboplastins. The variance in the PT ratios among the five thromboplastins was compared with the variance obtained with the INR values derived from the PT ratios when using the international sensitivity indexes provided by the manufacturer. Our results indicate that, even during the induction phase, there is less variance with the INR system than with the PT ratio system.
Anticoagulants↗
Evaluation of a soluble fibrin assay in patients with suspected pulmonary embolism.
In order to determine the clinical utility of an enzyme immunoassay (EIA) for soluble fibrin in patients with suspected pulmonary embolism (PE), 195 unselected patients with suspected PE underwent blood sampling for measurement of plasma levels of soluble fibrin, and objective testing for PE. A soluble fibrin result of < or = 0.75 micrograms/ml showed a sensitivity of 100% for PE and a specificity of 12.8%, whereas a soluble fibrin result of < or = 1.35 micrograms/ml showed a sensitivity of 90.3% and a specificity of 49.4% for PE. The soluble fibrin assay has potential clinical utility in excluding PE.
Adolescent↗
Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals.
Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.
Bacterial Proteins↗
Freeze trapping of reaction intermediates.
Structural intermediates in a biological reaction may be monitored by rapid spectroscopic or somewhat slower structural techniques. Intermediates may evolve in real time (no trapping), be stabilized by chemical manipulation of the reactants, the macromolecule or the solvent (chemical trapping), or be stabilized by lowering the temperature (freeze trapping). The last is beginning to be coupled with X-ray diffraction, electron cryomicroscopy and solid-state NMR approaches to characterize the trapped intermediates. Care in conducting such experiments, together with an awareness of possible artefacts, is essential if reliable structural results are to be obtained.
Cryopreservation↗
X-ray crystallography at extremely low temperatures.
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Carbon Monoxide↗
Evaluation of a soluble fibrin assay in patients with suspected deep vein thrombosis.
BACKGROUND: The objective of this study was to determine the clinical utility of an enzyme immunoassay (EIA) for soluble fibrin in patients with clinically suspected deep vein thrombosis (DVT). METHODS AND RESULTS: 101 unselected patients with clinically suspected DVT underwent blood sampling for measurement of plasma levels of soluble fibrin, and objective testing for DVT. According to results of the objective tests, patients were classified as DVT-positive (n = 34) or DVT-negative (n = 67). Using different cut-points of soluble fibrin results, the sensitivities, specificities, positive and negative predictive values of the soluble fibrin assay were calculated. A soluble fibrin result of < or = 0.75 mg/ml showed a sensitivity and negative predictive value of 100%, and a specificity of 17.9% for DVT, a soluble fibrin result of < or = 1.40 mg/ml showed a sensitivity of 91.2% and a negative predictive value of 93.6%, and a specificity of 65.7% for DVT, whereas a soluble fibrin result of < or = 8.0 mg/ml showed a specificity and positive predictive value of 100% for DVT. CONCLUSIONS: This study demonstrates that the soluble fibrin assay used in the study has potential clinical utility as a diagnostic test in patients with clinically suspected DVT and supports further evaluation of this assay.
Evaluation Studies as Topic↗
Laue crystallography for studying rapid reactions.
Precise quantitation of static and time-resolved Laue diffraction patterns is undeniably more complex than for monochromatic patterns. Recent advances in integration and scaling algorithms demonstrate that, with suitable care in the conduct of the Laue experiment itself, Laue data sets can be obtained which rival the best monochromatic data sets in accuracy and completeness. These algorithms deal in an integrated fashion with the several main problems of Laue diffraction patterns: the elongated spots which arise from mosaic crystals, the spatial overlaps which occur in crowded diffraction patterns, the energy overlaps which arise from the mapping of a central line in reciprocal space onto a single spot in detector space, and wavelength normalization.
Journal Article↗
Photolysis-induced structural changes in single crystals of carbonmonoxy myoglobin at 40 K.
Myoglobin's reversible binding of oxygen is a model for studies of protein control of ligand binding and discrimination. Protein relaxation and geminate ligand rebinding subsequent to ligand photodissociation have been studied extensively by a variety of techniques. The ps to ns time scales for these processes are still much shorter than the ms time resolution of X-ray diffraction experiments, but it may be possible to trap these intermediates at low temperatures. We report here an X-ray diffraction investigation of structural changes induced by photolysis of carbonmonoxy myoglobin crystals at 40 K. Our results provide a structural basis for the interpretation of ambient and low temperature spectroscopic observations and molecular dynamics simulations of the ligand photodissociation and binding processes in haem proteins.
Animals↗
Dye yellow vs. argon green laser in panretinal photocoagulation for proliferative diabetic retinopathy: a comparison of minimum power requirements.
We compared the power required to achieve a retinal burn with the dye yellow laser (wavelength 577 nm) and the argon green laser (wavelength 514 nm) in 49 eyes of 38 patients with proliferative diabetic retinopathy who underwent panretinal photocoagulation. All eyes were treated with both lasers. The dye laser required on average 35.7% less power to achieve a retinal burn than the argon laser (p = 0.0001). With both lasers more power was needed in patients older than 55 years than in those aged 55 years or less and in patients with light skin versus those with dark skin (p less than 0.02). The lower power required with the dye yellow laser allows facilitated photocoagulation in patients in whom treatment with the argon green laser would be problematic.
Adult↗