Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex.

Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into VL and VH as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1.

Nitroxide- and Cu2+-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important α-helix of the human glutathione S-transferase A1. Distance measurements on various spin-labeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu2+-based ESR distance measurements. Cu2+-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.

Dual Lifetimes for Complexes between Glutathione-S-transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging.

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products.

BACKGROUND: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. OBJECTIVE: To isolate and characterize the FVIIIFT fraction in rFVIII products. METHODS: Protein fractions unable (FVIIIFT ) and able (FVIIIEL ) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. RESULTS AND CONCLUSIONS: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL , FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Eliminating Spurious Zero-Efficiency FRET States in Diffusion-Based Single-Molecule Confocal Microscopy.

Single-molecule Förster resonance energy transfer (smFRET) of freely diffusing biomolecules using confocal microscopy is a simple and powerful technique for measuring conformation and dynamics. However, a spurious zero-FRET population can significantly distort the measured histograms and lead to incorrect results, particularly in measurements of intrinsically low-FRET systems. Using a model system consisting of duplex DNAs, we show that there are two important contributions to the zero-FRET state: (1) formation of a dark triplet state of the acceptor dye and (2) the presence of donor-only strands due to incomplete hybridization between donor- and acceptor-labeled strands. The combined strategy of using Trolox as a triplet-state quencher and labeling the same DNA strand with donor and acceptor dyes effectively eliminates the zero-FRET population, even for constructs with intrinsically low FRET efficiencies. This strategy allows us to perform smFRET experiments using a simple confocal microscope with improved accuracy.