| AUTHOR |
TITLE |
Eric A. Johnson,
Yoav Evron,
Richard E. McCarty |
Resonance Energy Transfer between Tryptophan 57 in the Subunit and Pyrene Maleimide Labeled Subunit of the Chloroplast ATP Synthase
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Materials and Methods
Resonance Energy Transfer Measurements. Fluorescence measurements were performed using either an Olis modified SLM Aminco-SPF-500C or a Shimadzu RF-5000 spectrofluorometer. All fluorescence measurements were performed at an excitation of 295 nm with the fluorescence emission monitored at 343 nm. Corrections were made in the fluorescence measurements for background, as measured by similarly treated CF1(εW15/57F), and protein content. The transfer efficiency (E) between tryptophan and pyrene was calculated based upon the fluorescence at 343 nm in the presence and absence of the pyrene acceptor molecule according to eq 2 where FD is the fluorescence of the donor (unlabeled CF1) and FDA is the fluorescence of the donor in the presence of the acceptor (labeled CF1). |
Carrie Hiser,
Denise A. Mills,
Michael Schall,
Shelagh Ferguson-Miller |
C-Terminal Truncation and Histidine-Tagging of Cytochrome c Oxidase Subunit II Reveals the Native Processing Site, Shows Involvement of the C-Terminus in Cytochrome c Binding, and Improves the Assay for Proton Pumping
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Materials and Methods
Proton Pumping Measurements. Proton pumping of the COVs was measured in a rapid scanning stopped-flow spectrophotometer (Olis RSM)....The cytochrome c oxidation rates were from Global fitting analysis by the Olis software to a single component exponential and were similar to those obtained from the kinetic traces at 550 nm, but the latter were perturbed by the influence of the phenol red absorbance and better fits were obtained with Global analysis to eliminate the dye contribution. |
Didier Nurizzo,
Heather M. Baker,
Qing-Yu He,
Ross T. A. MacGillivray,
Anne B. Mason,
Robert C. Woodworth,
Edward N. Baker. |
Crystal Structures and Iron Release Properties of Mutants (K206A and K296A) That Abolish the Dilysine Interaction in the N-Lobe of Human Transferrin.
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Kinetics of Iron Release. The kinetics of iron removal from the mutant proteins, and the processing of the kinetic data, were carried out as described previously (18,21). Tiron, at a concentration of 12 mM, was used as the chelator for iron removal at pH 7.4 (in 50 mM Hepes), and EDTA, at a concentration of 4 mM, was used as the chelator at pH 5.6 (in 50 mM Mes). Using these high concentrations of high-affinity, nonsynergistic chelators should preclude any significant back-reaction. The reactions were monitored by following the absorbance increase at 480 nm [for Fe(III)-Tiron] or the absorbance decrease at 293 or 470 nm (for iron removal by EDTA), with a protein concentration of ~40 µM. For the K206A and K296A mutants at pH 5.6, and for the K206A/K296A double mutant at pH 7.4, iron release was very slow, and the rate constants were estimated from a 5 h assay, carried out in duplicate, using the Ainf value from the wild type reaction for curve fitting. For the wild type hTf/2N at pH 7.4 and the K206A/K296A double mutant at pH 5.6, the rate of release was moderate, and data were collected for at least four half-lives, resulting in almost complete iron removal. These experiments were carried out using a Cary 219 spectrophotometer under the control of the OLIS-219s program. For the wild type hTf/2N at pH 5.6, release was very rapid and the kinetics were analyzed using an OLIS-RSM 1000 stopped-flow spectrophotometer which is able to record the spectral change in real time. The wavelengths cited above were used as the central wavelength in each assay, with a protein concentration of ~6.5 µM. All measurements were taken at 25 °C.
Iron Retention Level as a Function of pH. Profiles showing the iron retention level as a function of pH were obtained as described previously. For each mutant, the fully Fe(III)-loaded protein was incubated against a series of buffers, chosen to give appropriate buffering over the pH range of 7.7-4.2. Each solution was maintained at 4 °C for a period of 1 week to achieve equilibrium. The percentage saturation with iron was then estimated from the visible absorption spectrum, by comparing its absorbance at the visible absorption maximum (458 nm for K206A and 469 nm for K296A) with that of the fully iron saturated protein. All UV-visible spectra were recorded on a Cary 219 spectrophotometer under the control of the OLIS-219s program (On-line Instrument Systems Inc., Bogart, GA). |
Igor Efimov,
Ciarán N. Cronin,
William S. McIntire |
Effects of Noncovalent FAD Binding on the Redox and CAtalytic Properties of p-Cresol Methyllhydroxylase
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Materials and Methods
Stopped-Flow Kinetic Experiments. The OLIS RSM-1000 rapid spectrum scanning stopped-flow apparatus (On-Line Instrument Systems, Inc., Bogart, GA) was used in the wavelength range from 350 to 650 nm, at 25 or 6 °C in aerobic solutions. Spectra were recorded each millisecond for a total of 4000 ms. Spectra and kinetic constants were computed by using the OLIS Factor Analysis software. |
Motohiro Horiuchi,
Gerald S. Baron,
Liang-Wen Xiong,
Byron Caughey |
Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain
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Experimental Procedures
Circular Dichroism Spectroscopy
Synthetic peptides were dissolved in deionized water to make 5 mg/ml stock solutions. Peptide solutions (40 mL) were combined with 160 mL of conversion buffer (200 mM KCl, 5 mM MgCl 2 , 50 mM citrate, pH 6.0) with or without 0.1% sarkosyl to give final peptide concentrations of 1 mg/ml . CD measurements were performed using a 0.1 mm path length quartz cylindrical cell with an OLIS-16 DSM CD Spectrophotometer. The following parameters were used: 0.05 nm monochromator resolution, 1 nm bandwidth, dual beam mode, 400 kHz sampling rate, 2 V high volts criterion. Wavelength calibration was done with (1S)-(+)-10 camphorsulfonic acid (Sigma). The temperature of sample chamber was held at 37 °C. Data were collected from 260 to 90 nm with 1 datum/nm. The resulting spectra were obtained by averaging 6 scans and subtracting spectra of the buffer with or without 0.1% sarkosyl. |
Aya Tanatani,
Matthew J. Mio,
Jeffrey S. Moore |
Chain Length-Dependent Affinity of Helical Foldamers for a Rodlike Guest
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Experimental Procedures
Spectroscopic Measurements. Circular Dichroism (CD) absorption spectroscopy measurements were recorded with a µM concentration range in 1 cm quartz cell on an On-Line Instruments Systems (OLIS) Cary 17 Conversion spectrophotometer. |
Javier Seraballi,
Kenneth L. Brown,
Stephen W. Ragsdale |
Acetyl Coenzyme A Synthesis from Unnatural Methylated Corrinoids: Requirement for "Base-Off" Coordination at Cobalt
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Acknowledgment
Footnote 18
...Initial rates were measured at 55 °C in a Cary UV-visible spectrophotometer modified by OLIS Instruments (Bogart, GA) using anaerobic 1 and 0.2 cm path length quartz cuvettes. All experiments were carried out under 1 atm of 100% CO (g). Initial rates were determined either by the initial slopes or by fits of the entire time courses to a double-exponential function followed by extrapolation to t = 0, with software provided by OLIS Instruments. |
Doug Barrick,
Nancy T. Ho,
Virgil Simplaceanu,
Chien Ho |
Distal Ligand Reactivity and Quaternary Structure Studies of Proximally Detached Hemoglobins/b>
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| Measurement of Rates of CO Association and Dissociation. Progress curves for CO binding and dissociation were determined by stopped-flow methods using an Olis stopped-flow apparatus (Bogart, Georgia). The dead-time of the instrument was around 3 ms. Binding and dissociation of CO was determined by measuring absorbance changes of the heme chromophore. Proteins were buffered at pH 7.0 using 0.1 M sodium phosphate and contained 10 mM imd. Buffers were purged extensively with argon to remove traces of oxygen. All reactions were measured at 20 °C. |
Valerie V. Smirnov,
Eric K. Woller,
Dereck Tatman,
Stephen G. Dimagno |
Structure and Photophysics of &neta;-Octafluoro-meso-tetraarylporphyrins
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Experimental Section
Photophysical Studies. Absorption spectra were obtained using an Olis modification of an Cary-14 UV-Vis-NIR spectrometer. |
James T. Trent III,
Angela N. Hvitved,
Mark S. Hargrove |
A Model for Ligand Binding to Hexacoordinate Hemoglobins
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Materials and Methods
Kinetic Experiments. Rapid mixing experiments were carried out with a Durrum stopped-flow apparatus interfaced with a personal computer using previously described methods (16, 17). Absorbance spectra were collected during CO binding using an On Line Instruments (OLIS) rapid scanning monochromator (RSM) stopped-flow apparatus. |
Susanna Herold,
Michael Exner,
Thomas Nauser |
Kinetic and Mechanistic Studies of the NO*-Mediated Oxidation of Oxymyoglobin and Oxyhemoglobin
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Experimental Procedures
Stopped-Flow Kinetic Analysis. Kinetic studies were carried out with an Applied Photophysics SX17MV single-wavelength stopped-flow instrument and an On-Line Instrument Systems, Inc. stopped-flow instrument equipped with an OLIS RSM 1000 rapid scanning monochromator. The width of the cells into the two spectrophotometers was 1 and 2 cm, respectively. The mixing time of the instruments was 2-4 ms. |
Justine P. Roth,
Jeffrey C. Yoder,
Tae-Jin Won,
James M. Mayer |
Application of the Marcus Cross Relation to Hydrogen Atom Transfer Reactions
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| The reactions were carried out in air-free acetonitrile solutions, ~10-4 M in metal complex and under psuedo - first-order or mixed second-order conditions, with an Olis stopped flow apparatus equipped with a rapid scanning optical spectrophotometer. |
Alexei A. Neverov,
R. S. Brown |
La3+-Catalyzed Methanolysis of Phosphate Diesters. Remarkable Rate Acceleration of the Methanolysis of Diphenyl Phosphate, Methyl p-Nitrophenyl Phosphate, and Bis(p-nitrophenyl) Phosphate.
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Experimental Section
Kinetic Measurements. The rates of methanolysis of 3 and 5 in buffered methanol solutions were followed by monitoring either the appearance of p-nitrophenolate at 400 nm or the complex of p-nitrophenolate with La3+ at 355 nm or by monitoring the disappearance of starting material at 285 nm with an OLIS modified Cary 17 UV-vis spectrophotometer at 25.0 ± 0.1 °C. |
Chia-Hui Tai,
Peter Burkhard,
David Gani,
Thierry Jenn,
Corey Johnson,
Paul F. Cook |
Characterization of the Allosteric Anion-Binding Site of O-Acetylserine Sulfhydrylase
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Methods and Materials
Presteady-State Kinetic Studies. Single-wavelength absorbance stopped-flow studies were carried out using an OLIS RSM stopped-flow spectrophotometer. Single-wavelength time courses were fitted using the software provided with an equation of the following general form. |
Irina Pozdnyakova,
Pernilla Wittung-Stafshede |
Copper Binding before Polypeptide Folding Speeds Up Formation of Active (Holo) Pseudomonas aeruginosa Azurin
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Experimental Section
Equilibrium Studies. GuHCl- induced equilibrium unfolding of apo-azurin was monitored by far-UV circular dichroism (OLIS Inc. instrument; 200-300 nm, 1mm cell) and tryotophan fluorescence. All experiments were performed in 100 mM Tris-HCl buffer (pH 7.2, 20 °C). There was no protein-concentration dependence for the unfolding transition (in the range 2-100 mM), the two spectroscopic methods yielded identical results, and the reaction was fully reversible. |
