A spin label study of egg white avidin.

Chignell, Colin F.; Starkweather, Donnas K.; Sinha, Birandra K.

doi:10.1016/s0021-9258(19)41225-8

Cited by 37 publications

(13 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To account for the apparent 6.5 G doublet hyperfine interaction, the geometry of one of the 0-hydrogens of the cysteine must be ~90°with respect to the orbital of the unpaired electron on the nitrogen such that its coupling constant is very small. An alternative explanation for the putative doublet splitting must also be considered based on nitroxide spin labeling studies of Chignell et al (1975) with egg white avidin. These workers observed that each of the three hyperfine lines due to the nitroxide were split into doublets, similar to the doublet we have attributed to hyperfine interaction with a proton.…”

Section: Resultsmentioning

confidence: 99%

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate

Salowe¹,

Bollinger²,

Ator³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, and catalyzes the conversion of nucleotides to deoxynucleotides. The mechanism of inactivation of RDPR by 2'-azido-2'-deoxynucleoside 5'-diphosphate (N3UDP) has been examined using a variety of isotopically labeled derivatives: (1'-, 2'-, 3'-, or 4'-[2H])-N3UDPs and 2'-[15N3, 13C]-N3UDP. Electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopy studies using these compounds indicate that the 2' carbon-nitrogen bond to the azide moiety is cleaved prior to or upon formation of the nitrogen-centered radical derived from the azide moiety of N3UDP. EPR studies reveal no hyperfine interactions of the nitrogen-centered radical with the 1', 2', 3', or 4' hydrogens of N3UDP. ESEEM studies however, reveal that the 1' and 4' deuterons are 3.3 +/- 0.2 and 2.6 +/- 0.3 A, respectively, from the nitrogen-centered radical. Further support for carbon-nitrogen bond cleavage is derived from studies of the interaction of oxidized R1, C225SR1, and C462SR1 with R2 and N3UDP. In all three cases, in contrast to the results with the wild type R1, azide is detected. Nitrogen-centered radical is not observed with either oxidized R1 or C225SR1 but is observed with C462SR1. These results suggest that C225 is required for the conversion of azide into N2 and a nitrogen-centered radical. The dynamics of the inactivation of RDPR by N3UDP have also been examined. Use of [3'-2H]N3UDP reveals an isotope effect of approximately 2 on the loss of the tyrosyl radical and the rate of inactivation of RDPR. In both cases the kinetics are complex, suggesting multiple modes of inactivation. In addition, several modes of inactivation are required to explain the observation that loss of the tyrosyl radical is slower than the rate of inactivation. Studies using [5'-3H]N3UDP reveal that the rapid inactivation is the result of the formation of a tight noncovalent complex between modified nucleotide, nitrogen-centered radical and RDPR. Destruction of the nitrogen-centered radical is a slow process which appears to be accompanied by decomposition of the modified nucleotide into PPi, uracil, and 2-methylene-3(2H)-furanone. The latter covalently modifies R1 and ultimately leads to loss of approximately 50% of the activity of R1.

show abstract

Section: Resultsmentioning

confidence: 99%

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate

Salowe¹,

Bollinger²,

Ator³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Although the coupling of bulky substituents to biotin has been shown to reduce the strength and stability of the biotin/ streptavidin bond, 19,21,28,29 the facile dissociation associated with † University of New Mexico School of Medicine. ‡ University of New Mexico.…”

Section: Introductionmentioning

confidence: 99%

“…Although the coupling of bulky substituents to biotin has been shown to reduce the strength and stability of the biotin/streptavidin bond, ,,, the facile dissociation associated with short time incubations is in sharp contrast to the typical stability associated with this system. The dissociation rate constant of the complex was determined to be as high as 0.05 s -1 at early times after binding, and to decrease with contact time.…”

Section: Introductionmentioning

confidence: 99%

Ligand Receptor Dynamics at Streptavidin-Coated Particle Surfaces: A Flow Cytometric and Spectrofluorimetric Study

et al. 1999

View full text Add to dashboard Cite

We have the studied the binding of 5- ((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 µm diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. We have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. We estimate the site number to be ≈1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is ≈(1.3 ( 0.3) × 10 7 M -1 s -1 . The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s -1 shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

show abstract

“…This k ads rate is moderately slower than that of free streptavidin–biotin in the bulk, likely because the biontinylated origami was first immobilized on mica, which limits the freedom of movement of the biotin and the accessibility of SA to biotin. It is also possible that the linking tether, which is bonded to biotin at biotin’s terminal carboxylic acid functional site, has reduced the biotin’s ability to hydrogen bond inside the streptavidin binding pocket. , The measured binding rate is much faster than that reported for SA-Qdots binding onto biotinylated DNA origami in solution . The mass and volume of the Qdot could decrease the mobility of the SA-Qdot complexes as compared to SA alone, or hydrodynamic forces may pull the Qdot/SA from the DNA origami, removing the biotin tether from the construct, resulting in an increased off time.…”

mentioning

confidence: 90%

Origami Arrays as Substrates for the Determination of Reaction Kinetics Using High-Speed Atomic Force Microscopy

et al. 2017

View full text Add to dashboard Cite

DNA nanostructures (DN) are powerful platforms for the programmable assembly of nanomaterials. As applications for DN both as a structural material and as a support for functional biomolecular sensing systems develop, methods enabling the determination of reaction kinetics in real time become increasingly important. In this report, we present a study of the kinetics of streptavidin binding onto biotinylated DN constructs enabled by these planar structures. High-speed AFM was employed at a 2.5 frame/s rate to evaluate the kinetics and indicates that the binding fully saturates in less than 60 s. When the the data was fitted with an adsorption-limited kinetic model, a forward rate constant of 5.03 × 10 s was found.

show abstract

A spin label study of egg white avidin.

Cited by 37 publications

References 15 publications

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate

Alternative model for mechanism-based inhibition of Escherichia coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine 5'-diphosphate

Ligand Receptor Dynamics at Streptavidin-Coated Particle Surfaces: A Flow Cytometric and Spectrofluorimetric Study

Origami Arrays as Substrates for the Determination of Reaction Kinetics Using High-Speed Atomic Force Microscopy

Contact Info

Product

Resources

About