Streptavidin crystals

Membrane proteins comprise another important class of protein crystallized in 2D. These proteins perform important functions as membrane channels and recognition sites for cells. Unlike the streptavidin crystals, membrane proteins... 

Relini A, Sottini S, Zuccotti S, et al. Measurement of the surface free energy of streptavidin crystals by atomic force microscopy. Langmuir 2003 19(7) 2908-2912. 

While a number of proteins have been crystallized in this manner, the majority of studies have focused on a robust system comprising the tetrameric protein streptavidin and the vitamin biotin. The choice of this system is primcirily motivated by the strong bond between biotin and streptavidin (having an association equilibrium constant, Ka Tbe binding properties were recently... 

Fig. XV-5. Fluorescence micrographs illustrating morphologies of two-dimensional (2D) sireptavidin crystals at three streptavidin/avidin ratios 15/85, 25/75, 40/60 from left to ri t. Scale bar is 100 gm (From Ref. 31.)...

Fig. 14 A schematic illustration of an orientated immobilization strategy via crystallized S-layer proteins a side view of the crystalline ordered S-layer proteins fused with the recognition center streptavidin for biotinylated biomolecules probes b front view of the orientated and crystalline ordered S-layer proteins...

Hendrickson, W. A., et al. (1989). Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. Proc. Natl. Acad. Sci. USA 86, 2190-2194. 

The availability of a new version of isotopically labeled ATP, [33P]ATP, provided benefits of safety and longer half-life. The lowered energy was also better suited for scintillation proximity assays (SPAs). The SPA was a major step forward because it eliminated the need for wash steps by capturing the [33P]-labeled peptide on a functionalized scintillating crystal, usually via a biotin-streptavidin interaction. 

The existence of -barrels was established for chymotrypsin at a very early stage in the now common protein crystal structure analyses. This enzyme contains two distorted six-stranded -barrels with identical topologies (Birktoft and Blow, 1972). A selection of -barrels in water-soluble proteins is given in Table I. The very abundant TIM-barrel consisting of eight parallel /1-strands was also detected rather early (Banner et al., 1975). Additional eight-stranded /1-barrels of this group are those of streptavidin (Hendrickson et al., 1989) and of the lipocalins (Newcomer et al., 1984). 

Fig. 3.49 TM-AFM phase image of 2D crystal of streptavidin recorded in ambient conditions [105]...

Figure 21, Images (a,c) and Fourier transforms (b,d) of helical crystals of streptavidin formed on lipid tubules containing DODA-EOa-biotin (50). (a,c) Stain striations extend along the tubules. Protein densities are particularly visible at tube edges, corresponding to streptavidin molecules viewed edge-on. Scale bar 40 nm. (b,d) Distribution of Fourier transform amplitudes from the tubes shown in (a,c) corresponding to about 1700 streptavidin molecules. The fine spacing between layer lines indicates a helical repeat of 47 nm. Visible diffraction peaks extend up to 1.7 nm (arrowhead in (b)]. Reproduced from ref. 242 (Ringler et al., Chem. Eur. J. 1997, 3, 620) with permission ofWiley-VCH.

For further functionalization, the surface of the crystal is activated prior to covalent coupling with 200 pL of a solution of NHS 50 mM and ED AC 200 mM in water (see Note 2). After 5 min, the activating solution is replaced by streptavidin 200pg/mL in acetate buffer 10 mM, pH 5 for 20 min. The residual reacting sites are blocked with 200 pL of a solution... 

Figure 1. Current Nanoscale Optofluidic Sensor Arrays, (a) 3D rendering of the NOSA device, (b) 3D rendering after association of the corresponding antibody to the antigen immobilized resonator, (c) Experimental data illustrating the successful detection of 45 pg/ml of anti-streptavidin antibody. The blue trace shows the initial baseline spectrum corresponding to Fig. la where the first resonator is immobilized with streptavidin. The red trace shows the test spectra after the association of anti-streptavidin. (d) Finite difference time domain (FDTD) simulation of the steady state electric field distribution within the 1-D photonic crystal resonator at the resonant wavelength, (e) SEM image demonstrating the two-dimensional multiplexing capability of the NOSA architecture.

Fig. 2 a Immobilization procedure based on the streptavidin/biotin interaction. The probe to be immobilized is biotinylated and interacts with streptavidin covalently fixed onto the crystal, b Immobilization procedure based on the direct coupling of a thiolated probe onto the crystal... 

Probes specific for P35S were immobilized on 10 MHz quartz crystals coated with gold, employing both the mentioned immobilization procedures streptavidin/dextran-modified surfaces using a biotinylated probe and direct thiol coupling with 5 thiolated probes. 

In order to improve the reproducibihty both for the immobihzation of the receptor and for the interaction between the aptamer and Tat protein, the aptamer has been thermally treated before the immobihzation. This treatment can unfold the aptamer RNA strand making the biotin label at the 5 end available for the interaction with streptavidin on the crystal surface. Before the immobihzation, the biotinylated aptamer was heated at 90 °C for 1 min to unfold the RNA strand and then cooled in ice for 10 min in order to block the RNA in its unfolded structure [72]. Six different crystals were modified with this treated aptamer and the frequency shift due to the immobihzation step was recorded. An average value (n = 6) of 63 10 Hz was obtained, confirming the efficacy of the thermal treatment on the aptamer in significantly improving the reproducibihty of this step (CV% = 16). 

Later Ebersole et al. described a novel method for the immobilisation of biological species [69] based on the spontaneous formation of avidin and streptavidin monolayers onto gold. The monolayers formed irreversibly from aqueous solutions onto freshly formed gold surfaces of Pz crystals monolayers formed on aged gold surfaces showed lower activity. They demonstrated the use of this method using a DNA hybridisation assay and incorporated their AMISA technique to enhance the measured frequency change. 

Fig. 6 Changes in / and D measured at two harmonics ( = 3 and = 7) of a Si02-modified 5 MHz QCM crystal upon subsequent additions of biotin-containing lipid vesicles (i), streptavidin (ii), biotin-modified single stranded 15-mer DNA (iii), and fully complementary single stranded DNA (iv). Also shown at the top is a schematic representation of the sensing template, illustrating the Voigt-based representation used in the analysis of the data. Note that the DNA film was treated as one layer that undergoes a structural change dming hybridization...

Alkaline phosphatase-biotin and oligonucleotide-biotin (herpes simplex vims (HSV) DNA) immobilized onto gold and silver substrates and quartz crystal microbalances coated with avidin and streptavidin p-nitrophenyl phosphate and HSV, respectively [57]... 

These authors solved the crystal structure of a proteolysed fragment of streptavidin complexed with selenobiotin using the MAD technique based on the Karle (1980) analysis (section 9.4). Streptavidin is a protein of poorly understood function, probably involving antibiotic properties. This crystal contains two seleniums per 252 residues. The study supports the suggestion that biologically produced selenomethyionyl proteins could make the MAD method broadly applicable (Hendrickson (1985) and section 9.8). 

Figure9.17 Selenobiotinyl streptavidin study, (a) Anisotropy in anomalous scattering factors for selenium. A-C spectra (thick lines) measured with the electric vector E parallel with crystal axes a, b and c respectively. Theoretical values for elemental selenium are shown as thin lines. Values off"...

Guilbault recommended drying the antibody-modified crystal before and after reaction with the dissolved antigen [236]. A further enhancement of affinity and sensitivity can be achieved by means of the streptavidin-biotin afHnity system, and this seems even to open up the possibility of using piezoelectric affinity sensors in solution [237, 238],... 

Efficient immobilization of aptamers on surfaces is necessary for the construction of tongh, stable sensors and assay systems as one necessary step to overcome limitations for practical applications (Bini et al., 2007). Bini et al. (2007) have compared thrombin aptamers immobilized on a gold snrface by chemisorption (thiolated aptamer) and by biotin-streptavidin interaction (biotinylated aptamer carrying a linker) on a gold surface modified by a thiol-dextran-streptavidin layer. The linker-modified aptamer immobilized via streptavidin-biotin showed better reproducibility and sensitivity results for the quartz crystal sensor. Aptamers can be used for the functionalization of titanium-alloy surfaces (e.g., implant material, scaffolds) to enhance cell adhesion. The aptamers directed to osteoblasts are fixed electrochemically on the snrface of the alloy and promote cell adhesion (Gno et al., 2005, 2007). 

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