[12] Fluorescence anisotropy applied to biomolecular interactions

Jameson, David M.; Sawyer, William H.

doi:10.1016/0076-6879(95)46014-4

Cited by 170 publications

(118 citation statements)

References 28 publications

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“…Although it does not form any ion pairs or hydrogen bonds in either structure, in A6, the side chains of Asp 26 underneath Lys 66 could also be a factor, as could any subtle structural perturbations in the peptide-MHC molecule itself.…”

Section: Discussionmentioning

confidence: 99%

“…5). Transforming this data directly into the fraction bound versus the concentration of free peptide permits fitting to an independent site binding model (16,26). The resulting K D from analysis of six separate experiments is 51 Ϯ 20 nM for K66A and 1.7 Ϯ 0.4 nM for K66R, compared with the value of 18 Ϯ 1 nM observed for Tax-3K5Flc binding to wild-type HLA-A2 (16).…”

Section: Activation Thermodynamics For Peptide Dissociation From the Lysmentioning

confidence: 99%

“…To fit the binding data, we took advantage of the fact that anisotropy measures concentration ratios and provides direct information on the fraction bound and free (26). Anisotropy was converted into the fraction bound as a function of free peptide and fit to a multiple equal and independent site model varying the number of sites and the equilibrium binding constant (16).…”

Section: Methodsmentioning

confidence: 99%

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Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

Baxter¹,

Gagnon

Davis-Harrison³

et al. 2004

Journal of Biological Chemistry

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Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg 65 and Lys 66 and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg 65 -Lys 66 region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.Recognition of a peptide bound to a major histocompatibility complex protein (peptide-MHC) 1 by the ␣␤ T cell receptor (TCR) is necessary for the initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. TCRs bind peptide-MHC in a diagonal-to-orthogonal fashion, interacting with elements of both the peptide and the MHC (1-3). Numerous studies have probed the interfaces between TCRs and their ligands through the use of altered peptides or site-directed mutagenesis of the MHC (e.g. see . These studies have been useful in identifying hot spots within individual interfaces, as well as predicting the biophysical mechanisms by which T cell receptors bind their ligand (9).In our studies of TCR recognition of the human class I MHC HLA-A*0201 (referred to as HLA-A2) presenting the Tax peptide (sequence LLFGYPVYV; see Ref. 10), we identified a functional hot spot consisting of arginine 65 and lysine 66 on the HLA-A2 ␣1 helix (4). In a mutagenesis experiment involving 15 HLA-A2 amino acids contacted by the Tax-HLA-A2-specific ␣␤ T cell receptor A6, only two mutations, Arg 65 3 Ala (R65A) and Lys 66 3 Ala (K66A), resulted in significantly reduced T cell effector functions when the mutants were used to present the Tax peptide to T cells expressing the A6 receptor. Similar results were found with T cells expressing a different Tax-HLA-A2-specific TCR, B7. Lys 66 was also identified as a critical position influencing T cell reactivity in at least one other mutagenesis study of HLA-A2 (8).The general nature of the Arg 65 -Lys 66 hot spot in the recognition of Tax...

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Section: Discussionmentioning

confidence: 99%

Section: Activation Thermodynamics For Peptide Dissociation From the Lysmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

Baxter¹,

Gagnon

Davis-Harrison³

et al. 2004

Journal of Biological Chemistry

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“…Fluorescence anisotropy was measured with a Beacon 2000 fluorescence polarization system (Panvera, Madison, WI) using fluorescein excitation ( ex ϭ 490 nm) and emission ( em ϭ 535 nm) filters. Protein binding increases the measured anisotropy of the fluorescein-conjugated RNA, based on restriction of segmental motion and rotational correlation time (36 …”

Section: Rna-protein Binding Affinitymentioning

confidence: 99%

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies

Choi¹,

Lai²,

Fedic³

et al. 2014

Journal of Biological Chemistry

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Background: Insects generally express a single tristetraprolin family member, zinc finger proteins that promote mRNA decay. Results: The Drosophila protein, Tis11, can promote mRNA decay in cells, and its deficiency in flies results in accumulation of certain mRNAs. Conclusion: Tis11 deficiency in Drosophila results in increases of potential target transcripts. Significance: Tis11 can affect post-transcriptional gene expression in adult flies by regulating mRNA decay.

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“…The fluorescence anisotropy of a 10.5 M solution of rhodamine-labeled lysin (19) was measured after addition of varying amounts of VERL [1.5 g (1.5 pMol) to 15 g (15 pMol)] and after 1 min the anisotropy was measured. The change in anisotropy was plotted against the lysin:VERL molar ratio and the inflection point taken as the maximum binding stoichiometry (24).…”

Section: Affinity Purification Of Ve Receptor For Lysin (Verl)mentioning

confidence: 99%

The abalone egg vitelline envelope receptor for sperm lysin is a giant multivalent molecule

Swanson

Vacquier

1997

Proc. Natl. Acad. Sci. U.S.A.

114

149

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Abalone sperm lysin is a 16-kDa acrosomal protein, which nonenzymatically and species selectively creates a hole in the egg vitelline envelope (VE) through which the sperm passes to reach the egg cell membrane. The crystal structures of both monomeric and dimeric lysins have been solved and the sequences of lysins from 20 abalone species have been determined. As a first step in understanding the molecular mechanism by which lysin creates a hole in the VE, its VE receptor was isolated. The VE receptor for lysin (VERL) is an unbranched, rod-like molecule with an approximate relative molecular mass of 2 million; half the mass being carbohydrate. Fluorescence polarization studies showed positive cooperativity in the binding of lysin to VERL (EC 50 Ϸ9 nM) and were consistent with the species selectivity of lysin in dissolving VEs. Each molecule of VERL bound between 126 and 142 molecules of monomeric lysin (two independent assays), showing that VERL possesses repetitive lysin-binding motifs.Many marine invertebrate species spawn gametes into seawater, where fertilization and embryogenesis occur. In sea urchins (1, 2) and abalone (3, 4), several closely related, congeneric species may have overlapping breeding seasons and habitats. Such species usually exhibit species selectivity (specificity) in experimental cross-fertilizations, meaning that fertilization occurs more readily between homospecific mixtures of sperm and eggs than between heterospecific mixtures. The demonstrated species selectivity in the recognition events between invertebrate sperm and egg is a valuable model bearing on the general problem of the biochemistry of molecular recognition between cells.In marine invertebrates, the study of gamete recognition proteins may also yield information on the mechanism by which species arise in the sea, where barriers to larval transport are not readily apparent (5). Evidence that gamete recognition proteins may be involved in speciation comes from analyses of nucleotide substitutions in sea urchin sperm bindins (1) and abalone sperm acrosomal proteins (4, 6). Such analyses show that the evolution of these proteins has been promoted by positive Darwinian selection, suggesting there is significant adaptive value in altering their primary structure.The availability of large quantities of marine invertebrate gametes permits a detailed characterization of their gamete surface molecules involved in fertilization. The abalone egg is surrounded by an elevated, glycoproteinaceous vitelline envelope (VE) 0.6 m in diameter (7,8). Spermatozoa bind to the VE, their acrosomal granule exocytoses, and lysin is released. Lysin creates a 3-m diameter hole in the VE through which the spermatozoan passes to reach the egg cell membrane. Electron micrographs of the lysin-created hole show it is composed of splayed-apart fibers of 13-nm diameter; covalent bonds are not broken when lysin disrupts the VE (7).Although gamete recognition proteins have been cloned from both mammals (9-13) and invertebrates (1-4, 6, 14-16), the molec...

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[12] Fluorescence anisotropy applied to biomolecular interactions

Cited by 170 publications

References 28 publications

Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies

The abalone egg vitelline envelope receptor for sperm lysin is a giant multivalent molecule

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