Dataset Information

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

ABSTRACT: The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.

SUBMITTER: Honegger A

PROVIDER: S-EPMC2253284 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

Honegger Annemarie A Spinelli Silvia S Cambillau Christian C Plückthun Andreas A

Protein science : a publication of the Protein Society 20051001 10

The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of expo ...[more]

PMID: 16195545

Similar Datasets

Project description:In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10-11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3-18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4-5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime.

Dataset Information

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

Publications

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets